Abstract
Our purpose was to characterize human dental pulp cells isolated by various methods and to examine the behavior of cells grown under various conditions for the purpose of pulp/dentin tissue engineering and regeneration. We compared the growth of human pulp cells isolated by either enzyme digestion or the outgrowth method. Expression of dentin sialophosphoprotein, Cbfa1, and two types of collagen (I and III) in these cells was examined by Western blot or reverse transcription/polymerase chain reaction. Growth of pulp cells on dentin and in collagen gel was also characterized. We found that different isolation methods give rise to different populations or lineages of pulp cells during in vitro passage based on their collagen gene expression patterns. Cells isolated by enzymedigestion had a higher proliferation rate than those isolated by outgrowth. Pulp cells did not proliferate or grew minimally on chemically and mechanically treated dentin surface and appeared to establish an odontoblast-like morphology with a cytoplasmic process extending into a dentinal tubule as revealed by scanning electron microscopy. The contraction of the collagen matrix caused by pulp cells was dramatic: down to 34% on day 14. Our data indicate that (1) the choice of the pulp cell isolation method may affect the distribution of the obtained cell populations, (2) a treated dentin surface might still promote odontoblast differentiation, and (3) a collagen matrix may not be a suitable scaffold for pulp tissue regeneration because of the marked contraction caused by pulp cells in the matrix. The present study thus provides important information and a basis for further investigations pre-requisite to establishing pulp tissue engineering/regeneration protocols.
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About I, Bottero MJ, Denato P de, Camps J, Franquin JC, Mitsiadis TA (2000) Human dentin production in vitro. Exp Cell Res 258:33–41
Alsberg E, Hill EE, Mooney DJ (2001) Craniofacial tissue engineering. Crit Rev Oral Biol Med 12:64–75
Batouli S, Miura M, Brahim J, T.W.Tsutsui, Fisher LW, Gronthos S, Robey PG, Shi S (2003) Comparison of stem-cell-mediated osteogenesis and dentinogenesis. J Dent Res 82:976–981
Bohl KS, Shon J, Rutherford B, Mooney DJ (1998) Role of synthetic extracellular matrix in development of engineered dental pulp. J Biomater Sci Polym Ed 9:749–764
Buurma B, Gu K, Rutherford RB (1999) Transplantation of human pulpal and gingival fibroblasts attached to synthetic scaffolds. Eur J Oral Sci 107:282–289
Carlson MA, Longaker MT (2004) The fibroblast-populated collagen matrix as a model of wound healing: a review of the evidence. Wound Repair Regen 12:134–147
Chan CP, Lan WH, Chang MC, Chen YJ, Lan WC, Chang HH, Jeng JH (2005) Effects of TGF-betas on the growth, collagen synthesis and collagen lattice contraction of human dental pulp fibroblasts in vitro. Arch Oral Biol 50:469–479
Chen S, Santos L, Wu Y, Vuong R, Gay I, Schulze J, Chuang HH, Macdougall M (2005) Altered gene expression in human cleidocranial dysplasia dental pulp cells. Arch Oral Biol 50:227–236
Couble ML, Farges JC, Bleicher F, Perrat–Mabillon B, Boudeulle M, Magloire H (2000) Odontoblast differentiation of human dental pulp cells in explant cultures. Calcif Tissue Int 66:129–138
Di Lenarda R, Cadenaro M, Sbaizero O (2000) Effectiveness of 1 mol L–1 citric acid and 15% EDTA irrigation on smear layer removal. Int Endod J 33:46–52
Drury JL, Mooney DJ (2003) Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 24:4337–4351
D’Souza R (2002) Development of the pulpodentin complex. In: Hargreaves KM, Goodis HE (eds) Seltzer and Bender’s Dental pulp. Quintessence, Carol Stream, pp 13–41
Froes JA, Horta HG, Silveira AB da (2000) Smear layer influence on the apical seal of four different obturation techniques. J Endod 26:351–354
Grinnell F (2000) Fibroblast-collagen-matrix contraction: growth-factor signalling and mechanical loading. Trends Cell Biol 10:362–365
Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, DenBesten P, Robey PG, Shi S (2002) Stem cell properties of human dental pulp stem cells. J Dent Res 81:531–535
Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 97:13625–13630
Haas VR, Santos AR, Jr, Wada ML (2001) Behaviour of fibroblastic cells cultured in collagen I using the sandwich technique. Cytobios 106 (Suppl 2):255–267
Hosoya S, Matsushima K, Ohbayashi E, Yamazaki M, Shibata Y, Abiko Y (1996) Stimulation of interleukin-1beta-independent interleukin-6 production in human dental pulp cells by lipopolysaccharide. Biochem Mol Med 59:138–143
Igarashi M, Irwin CR, Locke M, Mackenzie IC (2003) Construction of large area organotypical cultures of oral mucosa and skin. J Oral Pathol Med 32:422–430
Lee KY, Alsberg E, Mooney DJ (2001) Degradable and injectable poly(aldehyde guluronate) hydrogels for bone tissue engineering. J Biomed Mater Res 56:228–233
Lee KY, Mooney DJ (2001) Hydrogels for tissue engineering. Chem Rev 101:1869–1879
MacDougall M, Simmons D, Luan X, Nydegger J, Feng J, Gu TT (1997) Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4. Dentin phosphoprotein DNA sequence determination. J Biol Chem 272:835–842
Mooney DJ, Powell C, Piana J, Rutherford B (1996) Engineering dental pulp-like tissue in vitro. Biotechnol Prog 12:865–868
Nakakura–Ohshima K, Watanabe J, Kenmotsu S, Ohshima H (2003) Possible role of immunocompetent cells and the expression of heat shock protein-25 in the process of pulpal regeneration after tooth injury in rat molars. J Electron Microsc (Tokyo) 52:581–591
Nakao K, Itoh M, Tomita Y, Tomooka Y, Tsuji T (2004) FGF-2 potently induces both proliferation and DSP expression in collagen type I gel cultures of adult incisor immature pulp cells. Biochem Biophys Res Commun 325:1052–1059
Nakashima M (1991) Establishment of primary cultures of pulp cells from bovine permanent incisors. Arch Oral Biol 36:655–663
Nakashima M, Iohara K, Ishikawa M, Ito M, Tomokiyo A, Tanaka T, Akamine A (2004) Stimulation of reparative dentin formation by ex vivo gene therapy using dental pulp stem cells electrotransfected with growth/differentiation factor 11 (Gdf11). Hum Gene Ther 15:1045–1053
Ohshima H, Nakakura–Ohshima K, Takeuchi K, Hoshino M, Takano Y, Maeda T (2003) Pulpal regeneration after cavity preparation, with special reference to close spatio-relationships between odontoblasts and immunocompetent cells. Microsc Res Tech 60:483–490
Okazaki M, Yoshimura K, Suzuki Y, Harii K (2003) Effects of subepithelial fibroblasts on epithelial differentiation in human skin and oral mucosa: heterotypically recombined organotypic culture model. Plast Reconstr Surg 112:784–792
Okiji T (2002) Pulp as a connective tissue. In: Hargreaves KM, Goodis HE (eds) Seltzer and Bender’s Dental Pulp. Quintessence, Carol Stream, pp 95–123
Onishi T, Kinoshita S, Shintani S, Sobue S, Ooshima T (1999) Stimulation of proliferation and differentiation of dog dental pulp cells in serum-free culture medium by insulin-like growth factor. Arch Oral Biol 44:361–371
Park SH, Hsiao GY, Huang GT (2004) Role of substance P and calcitonin gene-related peptide in the regulation of interleukin-8 and monocyte chemotactic protein-1 expression in human dental pulp. Int Endod J 37:185–192
Patel T, Park SH, Lin LM, Chiappelli F, Huang GT (2003) Substance P induces interleukin-8 secretion from human dental pulp cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 96:478–485
Priam F, Ronco V, Locker M, Bourd K, Bonnefoix M, Duchene T, Bitard J, Wurtz T, Kellermann O, Goldberg M, Poliard A (2005) New cellular models for tracking the odontoblast phenotype. Arch Oral Biol 50:271–277
Saito T, Ogawa M, Hata Y, Bessho K (2004) Acceleration effect of human recombinant bone morphogenetic protein-2 on differentiation of human pulp cells into odontoblasts. J Endod 30:205–208
Schmalz G, Garhammer P, Schweiki H (1996) A commercially available cell culture device modified for dentin barrier tests. J Endod 22:249–252
Schmalz G, Schuster U, Thonemann B, Barth M, Esterbauer S (2001) Dentin barrier test with transfected bovine pulp-derived cells. J Endod 27:96–102
Shi S, Gronthos S (2003) Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 18:696–704
Smith AJ (2002) Dentin formation and repair. In: Hargreaves KM, Goodis HE (eds) Seltzer and Bender’’s Dental Pulp. Quintessence, Carol Stream, pp 41–63
Tsukamoto Y, Fukutani S, Shin–Ike T, Kubota T, Sato S, Suzuki Y, Mori M (1992) Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts. Arch Oral Biol 37:1045–1055
Vacanti CA, Langer R, Schloo B, Vacanti JP (1991) Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation. Plast Reconstr Surg 88:753–759
Vacanti JP (2003) Tissue and organ engineering: can we build intestine and vital organs? J Gastrointest Surg 7:831–835
Vernon RB, Sage EH (1996) Contraction of fibrillar type I collagen by endothelial cells: a study in vitro. J Cell Biochem 60:185–197
Vidic B, Chen R, Taylor JJ (1972) Functional behavior and structural modification of pulpal fibroblasts cultured in vitro. Archs Arch Anat Histol Embryol 55:51–73
Zhu YK, Umino T, Liu XD, Wang HJ, Romberger DJ, Spurzem JR, Rennard SI (2001) Contraction of fibroblast-containing collagen gels: initial collagen concentration regulates the degree of contraction and cell survival. In Vitro Cell Dev Biol Anim 37:10–16
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We greatly appreciated the technical assistance of Alicia Thompson (Center for Electron Microscopy and Microanalysis) with the SEM analysis.
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Most of this work was performed at UCLA School of Dentistry.
This study was supported in part by an Endodontic Research Grant from the American Association of Endodontists Foundation (G.T.-J.H.)
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Huang, G.TJ., Sonoyama, W., Chen, J. et al. In vitro characterization of human dental pulp cells: various isolation methods and culturing environments. Cell Tissue Res 324, 225–236 (2006). https://doi.org/10.1007/s00441-005-0117-9
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DOI: https://doi.org/10.1007/s00441-005-0117-9