Journal of Periodontal and Implant Science

Korean Academy of Periodontology (대한치주과학회)
권호 표지

Change of Stratification of Three Dimensional Culture by Gingival Keratinocytes & Fibroblasts

치은 각화상피세포와 섬유아세포를 이용한 삼차원적 배양시 중층화 동안의 변화

  • Jung, Tae-Heup (Department of Periodontology, School of Dentistry, Wonkwang University) ;
  • Hyun, Ha-Na (Department of Periodontology, School of Dentistry, Wonkwang University) ;
  • Kim, Yun-Sang (Department of Periodontology, School of Dentistry, Wonkwang University) ;
  • Kim, Eun-Cheol (Department of Oral Pathology, School of Dentistry, Wonkwang University) ;
  • You, Hyung-Keun (Department of Periodontology, School of Dentistry, Wonkwang University) ;
  • Shin, Hyung-Shik (Department of Periodontology, School of Dentistry, Wonkwang University)
  • 정태흡 (원광대학교 치과대학 치주과학교실) ;
  • 현하나 (원광대학교 치과대학 치주과학교실) ;
  • 김윤상 (원광대학교 치과대학 치주과학교실) ;
  • 김은철 (원광대학교 치과대학 구강병리학교실) ;
  • 유형근 (원광대학교 치과대학 치주과학교실) ;
  • 신형식 (원광대학교 치과대학 치주과학교실)
  • Published : 2002.03.30
Download PDF
⟨ Previous Next ⟩

Abstract

Epithelial-mesenchymal interaction plays a important role in cell growth and differentiation. This interaction is already well known to have an importance during the organ development as well as cell growth and differentiation. However, in vitro experimental model is not well developed to reproduce in vivo cellular microenvironment which provide a epithelial-mesenchymal interaction. Because conventional monolayer culture lacks epithelial-mensenchymal interaction, cultivated cells have an morphologic, biochemical, and functional characteristics differ from in vivo tissue. Moreover, it's condition is not able to induce cellular differention due to submerged culture condition. Therefore, the aims of this study were to develop and evaualte the in vitro experimental model that maintains epithelial-mesenchymal interaction by organotypic raft culture, and to characterize biologic properties of three-dimensionally reconstituted oral keratinocytes by histological and immunohistochemical analysis. The results were as follow; 1. Gingival keratinocytes reconstituted by three-dimensional organotypic culture revealed similar morphologic characteristics to biopsied patient specimen showing stratification, hyperkeratinosis, matutation of epithelial architecture. 2. Connective tissue structure was matured, and there is no difference during stratification period of epithelial 3-dimensional culture. 3. The longer of air-exposure culture on three-dimensionally reconstituted cells, the more epithelial maturation, increased epithelial thickness and surface keratinization 4. In reconstitued mucosa, the whole epidermis was positively stained by anti-involucrin antibody, and there is no difference according to air-exposured culture period. 5. The Hsp was expressed in the epithelial layer of three-dimensionally cultured cells, especially basal layer of epidermis. The change of Hsp expression was not significant by culture stratification. 6. Connexin 43, marker of cell-cell communication was revealed mild immunodeposition in reconstitued epithelium, and there is no significant expression change during stratification. These results suggest that three-dimensional oragnotypic co-culture of normal gingival keratinocytes with dermal equivalent consisting type I collagen and gingival fibroblasts results in similar morphologic and immunohistochemical characteristics to in vivo patient specimens. And this culture system seems to provide adequate micro-environment for in vitro tissue reconstitution. Therefore, further study will be focused to study of in vitro gingivitis model, development of novel perioodntal disease therapeutics and epithelial-mensenchymal interaction.

Keywords

References

  1. Carranza FAJr. Glickman's Clinical Periodontology. WB Saunders Company 7th Edi, 1990
  2. Hancock EB. Regeneration procedures. Proceedings of the world workshop in clinical periodontics. The American Academy of Periodontology. Discussion Section VI 1-26, 1989
  3. Caffesse RG:Resective procedures. Proceedings of the world workshop in clinical periodontics. The American Academy of Periodontology. Discussion Section VI 1-27, 1989
  4. Bowers GM, Schallhorn RG, Mellonig JT : Histologic evaluation of new attachment in human intrabony defect. A literature review. J Periodontol 53:509-514, 1982 https://doi.org/10.1902/jop.1982.53.8.509
  5. Melcher AH : Repair of wounds in the periodontium of the rat. Influence of periodontal ligament on osteogenesis. Achs Oral Biol 15: 1183-1204, 1970 https://doi.org/10.1016/0003-9969(70)90010-5
  6. Melcher AH : On the repair potential of periodontal tissues. J Periodontol 47: 256-260, 1976 https://doi.org/10.1902/jop.1976.47.5.256
  7. Boyko GA, Melcher AH, Brunette DM : Formation of new periodontal ligament by periodontal ligament cells implanted in vivo after culture in vitro. A preliminary study of transplanted roots in the dog. J Periodontol Res 16L 73-88, 1981
  8. Polson AM, Caton J : Factors influenceing periodontal repair and regeneration. J Periodontol 53: 617-625, 1982 https://doi.org/10.1902/jop.1982.53.10.617
  9. Isidor F, Karring T, Nyman S, Lindhe J : The significance of coronal growth of periodontal ligament tissue for new attachment formation. J Periodontol 13:145-150, 1986 https://doi.org/10.1111/j.1600-051X.1986.tb01448.x
  10. Arnold LF, Baram P : In vitro culture of periodontal ligament cells. J Dent Res 1:953-959, 1972
  11. Marmary Y, Brunette DM, Heersche JNM : Differences in vitro between cells derived from periodontal ligamnet and skin of Macaca Irus. Archs Oral Biol 21:709-716, 1976 https://doi.org/10.1016/0003-9969(76)90060-1
  12. Brunette DM, Melcher AH, Moe HK : Cultrue and origin of epithelium-like and fibroblast-like cells from porcine periodontal ligament explants and cell supension. Archs Oral Biol 21: 393-400, 1976 https://doi.org/10.1016/0003-9969(76)90001-7
  13. Brunette DM, Janoza RJ, Marmary Y, Chan J, Melcher AH : Interactions between epithelial and fibroblast-like cells in cultures derived from monkey periodontal ligament. J Cell Sci 27: 127-140, 1977
  14. Brunette DM, Heersche JNM, Purdon AD, Sodek J, Moe HK, Assuras JN :In vitro cultural parameters and protein and prostaglandin secretion of epithelial cells derived from porcine ress of malassez. Archs Oral Biol 24: 199-203, 1979 https://doi.org/10.1016/0003-9969(79)90140-7
  15. Blomlof I, Otteskog P : Composition of Human Periodontal Ligament Cells in tissue Culture. Scand. J Dent Res 89: 43-47, 1981
  16. Ragnarsson B, Carr G, Daniel JC : Isolation and growth of human perodontal ligametn cells in vitro. J Dent Res 64: 1026-1030, 1985 https://doi.org/10.1177/00220345850640080101
  17. Levine JF, Stockdale EF. Cell-cell interactions promote mammary epithelial cell differentiation. J Cell Biol 100: 1415-1422, 1985 https://doi.org/10.1083/jcb.100.5.1415
  18. Boyce ST, Hansbrough JF. Biologic attachment, growth, and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate. Surgery 103: 421-431, 1988
  19. Shahabeddin L, Berthod F, Damour O, Collombel C. Characterization of skin reconstructed on a chitosa-cross-linked collagen-glycosaminoglycan matrix. Skin Phamacol 3: 107-114, 1990 https://doi.org/10.1159/000210857
  20. Prunieras M. Recent advances in epidermal cell cultures. Arch Dermatol Res 264: 243-247, 1979 https://doi.org/10.1007/BF00431135
  21. Peault B. In-vitro models of stroma-dependent lymphopoiesis. Semi Immunol 7: 169-157, 1995 https://doi.org/10.1016/1044-5323(95)90044-6
  22. Sutherland RM. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science 240: 177-184, 1988 https://doi.org/10.1126/science.2451290
  23. Tiollier J, Dumas H, Tardy M, Tayot JL. Fibroblast behavior on gels of type I, III, and IV human placental collagens. Exp Cell Res 191: 95-104, 1990 https://doi.org/10.1016/0014-4827(90)90041-8
  24. Hoffman RM. Three-dimensional histoculture: origins and applications in cancer research. Cancer Cell 3: 86-92, 1991
  25. Robbins KT, Connors KM, Storniolo AM, Hanchett C, Hoffman RM. Sponge-gel supported histoculture drug response assay for head and neck cancer. Correlations with clinical response to cisplatin. Arch Otolaryngol Head Neck Surg 120: 288-292, 1994 https://doi.org/10.1001/archotol.1994.01880270036007
  26. Robbins KT, Varki NM, Storniolo AM, Hoffman H, Hoffman RM. Drug response of head and neck tumors in native-state histoculture. Arch Otolaryngol Head Neck Surg 117: 83-86, 1991 https://doi.org/10.1001/archotol.1991.01870130089022
  27. Tomakide P, Fusenig NE, Kohl A, Komosch. Histomorphological and biochemical differentiation capacity in organotypic co-culture of primary gingival cells. J Periodont Res 32:400, 1997
  28. Naraynan AS, Nakae H, Miki Y : Bioligy of the gingiva : The connective tissue in health and disease and molecular aspects of periodontal regeneration a reattachment. Recent Advances in Clinical Periodontology. Elsevier Science Publishers 51-61, 1988
  29. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331-343, 1975 https://doi.org/10.1016/S0092-8674(75)80001-8
  30. Mendelsohn MG, Dilorenzo TP, Abramson AL, Steinberg BM. Retinoic acid regulate in vitro. the two normal pathways of differentiation of human laryngeal keratinocytes. In Vitro Cell Dev Biol 27A: 137-141, 1991
  31. Bell E, Ivarsson B, Merrill C. Production of a tissue-like structure by contractio of collgen lattices by human fibroblast of different proliferative potential in vitro. Proc Natl Acad Sci USA 1979, 76; 1274-1279 https://doi.org/10.1073/pnas.76.3.1274
  32. Choi YS, and Fuchs E. TGF alpha induces collagen degradation and cell migration in differentiating human epidermal raft cultures. Cell Regul 2: 613-662, 1991
  33. Oda D, Savard CE, Eng L, Lee SP. The effect of N-methyl-N'-mitrosoguanidine (MNNG) on cultured dog pancreatic duct epitheial cells. Pancreas 12: 109-116, 1996 https://doi.org/10.1097/00006676-199603000-00001
  34. Otto WR, Nanchahal J, Lu QL, Boddy N, Dover R. Survival of allogenic cells in cultured organotypic skin grafts. Plastic Reconst Surg 96: 166-176, 1995 https://doi.org/10.1097/00006534-199507000-00025
  35. Garlick JA, Taichman LB. Fate of human keratinocytes during reepithelialization in an organotypic culture model. Lab Inv 70: 916-924, 1994
  36. Dotto GP, Moellmann G, Ghosh S, Edwards M, Halaban RJ. Cell Biol 109, 3115-3128. 1989 https://doi.org/10.1083/jcb.109.6.3115
  37. Vescio RA, Redfern CH, Nelson TJ. Ugoretz S, Stern PH, Hoffman RM. In vivo-like drug responses of human tumors growing in threedimensional, gel-supported, primary culture. Proc Natl Acad Sci 84: 5029-5033, 1987 https://doi.org/10.1073/pnas.84.14.5029
  38. Sarri Atula, Reidar Greman, Stina Syrjanen. Fibroblast can modulate the phenotype of malignant epithelial cells in vitro. Exp Cell Res 235: 180-87, 1997 https://doi.org/10.1006/excr.1997.3676
  39. Asselineau D, Bernard BA, Bailly C, Darmon M, Prunieras M. Human epidermis reconstructed by culture. Is it normal? J Invest Dermatol 86: 181-186, 1986 https://doi.org/10.1111/1523-1747.ep12284237
  40. Tavakkol A, Varani J, ELder JT, Zouboulis CC. Maintenace of human skin in organ culture: role for Insulin-like growth factor -1 receptor and EGFR. Arch Dermatol Res 291;643-651, 1999 https://doi.org/10.1007/s004030050469
  41. A new skin equivalent model: dermal substrate that combines de-epidermized dermis with fibroblasts-populated collagen matrix. J Dermatol Sci 23:132-137, 2000 https://doi.org/10.1016/S0923-1811(00)00068-2
  42. Ashburner M, Bonner JJ. The induction of gene activity in Drosophilia by heat strock. Cell 17:241-5, 1979 https://doi.org/10.1016/0092-8674(79)90150-8
  43. Burdon RH. Heat shock and the heat shock proteins. Biochem J 240:313-24, 1986 https://doi.org/10.1042/bj2400313
  44. Lindquist S. The heat-shock response. Annu Rev Biochem 55:1151-91, 1986 https://doi.org/10.1146/annurev.bi.55.070186.005443
  45. Pelham H. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell 46:959-61, 1986 https://doi.org/10.1016/0092-8674(86)90693-8
  46. Schlesinger MJ. Heat shock proteins: The search for functions. J Cell Biol. 103:321-5, 1986 https://doi.org/10.1083/jcb.103.2.321
  47. Muramatsu T., Tada H, Kobayashi N, et al. Induction of the 72-KD heat shock protein in organ-cultured normal human skin. J Invest Dermatol 98:786-90, 1992 https://doi.org/10.1111/1523-1747.ep12499953
  48. Polla BS. Heat (shock) and the skin. Dermatologica ;180:113-7, 1990 https://doi.org/10.1159/000248008
  49. Kusher DI, Ware CF, Gooding LR. Induction of the heat shock response protect cells from lysis by tumor necrosis foactor. J Immunol 145:2925-31, 1990
  50. Ciocca DR. Evidence for modulation of a 24K protein in human endometrium during the menstrual cycle. J Clin Endocrinol Metab 57:496-9, 1983 https://doi.org/10.1210/jcem-57-3-496
  51. Michael FP, Nancy A N-G, Amrtin B, Geoffrey LG. Estrogen receptor localization in the female genital tract. Am J Pathol 123:280-92, 1986
  52. Young D, Lathigra R, Hendrix R, Sweetser D. Young RA. Stress proteins are immune targets in leprosy and tuberculosis. Proc Natl Acad Sci USA 85:4276-70, 1988 https://doi.org/10.1073/pnas.85.12.4276
  53. lindquis S, Craig EA. The heat shock proteins. Ann Rev Genet 22:631-77, 1988 https://doi.org/10.1146/annurev.ge.22.120188.003215
  54. Sauchez ER, Meshinchi S, Tienrungroj W. Relationship of the 90k-Da murine heat shock protein to the untransformed and transformed states of the L cell glucocorticoid receptor. J Biol Chem 262:6989-91, 1987
  55. McCulloch CAG : Melcher AH : Cell density and cell generation in the periodontal ligament of mice. Am J Anat 167 : 43-58, 1993b https://doi.org/10.1002/aja.1001670105
  56. Polla BS. A role of heat shock proteins in inflammation?. Immunology Today 9:134-137, 1988 https://doi.org/10.1016/0167-5699(88)91199-1
  57. Burdon RJ: Heat shock and the heat shock proteins. Biochem J 240, 313-324
  58. Welch WJ, Suhan JP:Cellular and biochemical events in mammalian cells furing and after recovery from physiological stress. J Cell Biol 103, 2035-2052, 1986 https://doi.org/10.1083/jcb.103.5.2035
  59. Nagata K., Sga S. adn Yamada K. M. A major collagen-binding protein of chick embryo fibroblasts is a novel heat shock protein. J Cell Biol 103, 223-229, 1986 https://doi.org/10.1083/jcb.103.1.223
  60. Kurkinen M. A., Taylor A., Garrels J. I. and Hogan B. L. M. Cell surface-associated proteins which bind native type IV collagen or gelatin. J Biol Chem 259, 5915-5922, 1984
  61. Bowers W, Blaha M, Alkhyyat A, Sankovich J, Kohl J, Wong G, Patterson D. Artificial human skin: cytokine , prostaglandin, Hsp70 and histological response to heat exposure. J Dermatol Sci 20: 172-182, 1999 https://doi.org/10.1016/S0923-1811(98)00079-6
  62. Hamblin AS Lymphokines, p1-71. Inmale(ed), In Focus, Oxford 1988
  63. Makowski L. X-ray diffraction studies of gap junction structure. In: Advances in cell biology. Vol 2. Miller K, editor. Greenwich, CT:Jai Press,pp. 119-158, 1988
  64. Musil LS, Cunningham BA, Edelman GM, Goodenough DA. : Dirrerentia phosphorylation of the gap junction protein connexin 43 in junction communication-competent and-deficient cell lines. J Cell Biol 111:2077-2088, 1990 https://doi.org/10.1083/jcb.111.5.2077
  65. Tomaki P, Cheng H, Kohl A, Komposch G. Connexin 43 expression is downregulated in raft culture of human keratinocyte exprssing the human papilloma virus 16 E5 protein. Cell Tissue Res 301;323-327, 2000 https://doi.org/10.1007/s004410000231