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Effect of enamel matrix protein derivative on the attachment, proliferation, and viability of human SaOs2 osteoblasts on titanium implants

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Abstract

The purpose of the present study was to investigate the effects of an enamel matrix protein derivative (EMD) on attachment, proliferation, and viability of human SaOs2 osteoblasts on titanium implants. A total of 220 sand-blasted and acid-etched (SLA) titanium discs were placed into 24-well culture plates. Before cell inoculation, McCoy’s 5A medium (MCM) containing EMD at 25 µg/ml, 50 µg/ml, 100 µg/ml, and 200 µg/ml was added, and the culture plates were incubated for 30 min. As control, MCM alone was used. Human osteoblast-like cells (SaOs2) (2×104 cells, fourth passage) were suspended in MCM containing 1% penicillin/streptomycin and 10% fetal bovine serum and then inoculated into the well chambers. The medium was changed after 3 days without the addition of EMD. At days 1, 3, and 6, DNA content of the cells was assessed using the CyQuant cell proliferation assay kit, and mitochondrial activity of the cells was measured using a CellTiter-Glo luminescent cell viability assay. The presence of EMD on the titanium discs at days 1 and 6 was evaluated using immunofluorescence stain (IFS) by means of polyclonal antibodies against amelogenin. Additionally, cell morphology was investigated using scanning electron microscopy. Enamel matrix derivative at 25 µg/ml, 50 µg/ml, 100 µg/ml, and 200 µg/ml demonstrated similar increases in cell proliferation as the control medium at days 3 and 6 (P>0.05 between groups, respectively). Proliferation, however, appeared to be ameliorated with increasing EMD concentrations. At 25 µg/ml and 50 µg/ml, EMD also demonstrated an increase in cell viability similar to the control medium at days 3 and 6 (P>0.05 between groups, respectively), while EMD at 100 µg/ml and 200 µg/ml resulted in statistically significant higher increase in cell viability than in the control medium at day 6 (P<0.001 between groups, respectively). In all test groups, IFS at day 6 was markedly lower than at day 1. Scanning electron microscopy revealed comparable cell morphology in all groups. Within the limits of the present study, it was concluded that EMD enhanced cell proliferation and viability of human SaOs2 osteoblasts on SLA titanium implants in a concentration-dependent manner.

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References

  1. Ahmad M, McCarthy MB, Gronowicz G (1999) An in vitro model for mineralization of human osteoblast-like cells on implant materials. Biomaterials 20:211–220

    Article  CAS  PubMed  Google Scholar 

  2. Bowers KT, Keller JC, Randolph BA, Wick DG, Michaels CM (1992) Optimization of surface micromorphology for enhanced osteoblast responses in vitro. Int J Oral Maxillofac Implants 7:302–310

    CAS  PubMed  Google Scholar 

  3. Casati MZ, Sallum EA, Nociti FH Jr, Caffesse RG, Sallum AW (2002) Enamel matrix derivative and bone healing after guided bone regeneration in dehiscence-type defects around implants. A histomorphometric study in dogs. J Periodontol 73:789–796

    PubMed  Google Scholar 

  4. Chang YL, Stanford CM, Wefel JS, Keller JC (1999) Osteoblastic cell attachment to hydroxyapatite-coated implant surfaces in vitro. Int J Oral Maxillofac Implants 14:239–247

    CAS  PubMed  Google Scholar 

  5. Cree IA, Pazzagli M, Mini E, Mazzei T, Hunter EM, Sutherland LA, Pinzani P, Gerli A, Andreotti PE (1995) Methotrexate chemosensitivity by ATP luminescence in human leukemia cell lines and in breast cancer primary cultures: comparison of the TCA-100 assay with a clonogenic assay. Anticancer Drugs 6:398–404

    CAS  PubMed  Google Scholar 

  6. Crouch SP, Kozlowski R, Slater KJ, Fletcher J (1993) The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. J Immunol Methods 160:81–88

    Article  CAS  PubMed  Google Scholar 

  7. Davenport DR, Mailhot JM, Wataha JC, Billman MA, Sharawy MM, Shrout MK (2003) Effects of enamel matrix protein application on the viability, proliferation, and attachment of human periodontal ligament fibroblasts to diseased root surfaces in vitro. J Clin Periodontol 30:125–131

    Article  CAS  PubMed  Google Scholar 

  8. Fincham AG, Moradian-Oldak J, Simmer JP, Sarte P, Lau EC, Diekwisch T, Slavkin HC (1994) Self-assembly of a recombinant amelogenin protein generates supramolecular structures. J Struct Biol 112:103–109

    Article  CAS  PubMed  Google Scholar 

  9. Franke Stenport V, Johansson CB (2003) Enamel matrix derivative and titanium implants. An experimental pilot study in the rabbit. J Clin Periodontol 30:359–363

    PubMed  Google Scholar 

  10. Gestrelius S, Andersson C, Johansson AC, Persson E, Brodin A, Rydhag L, Hammarstrom L (1997) Formulation of enamel matrix derivative for surface coating. Kinetics and cell colonization. J Clin Periodontol 24:678–684

    CAS  PubMed  Google Scholar 

  11. Gestrelius S, Andersson C, Lidstrom D, Hammarstrom L, Somerman M (1997) In vitro studies on periodontal ligament cells and enamel matrix derivative. J Clin Periodontol 24:685–692

    CAS  PubMed  Google Scholar 

  12. Hammarström L, Heijl L, Gestrelius S (1997) Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins. J Clin Periodontol 24:669–677

    PubMed  Google Scholar 

  13. Heijl L, Heden G, Svardstrom G, Ostgren A (1997) Enamel matrix derivative (Emdogain) in the treatment of intrabony periodontal defects. J Clin Periodontol 24:705–714

    CAS  PubMed  Google Scholar 

  14. Jiang J, Safavi KE, Spangberg LS, Zhu Q (2001) Enamel matrix derivative prolongs primary osteoblast growth. J Endod 27:110–112

    Article  CAS  PubMed  Google Scholar 

  15. Kohn DH, Sarmadi M, Helman JI, Krebsbach PH (2002) Effects of pH on human bone marrow stromal cells in vitro: implications for tissue engineering of bone. J Biomed Mater Res 60:292–299

    Article  CAS  PubMed  Google Scholar 

  16. Lindskog S, Hammarström L (1982) Formation of intermediate cementum. III: 3H-tryptophan and 3H-proline uptake into the epithelial root sheath of Hertwig in vitro. J Craniofac Genet Dev Biol 2:171–177

    CAS  PubMed  Google Scholar 

  17. Lyngstadaas SP, Lundberg E, Ekdahl H, Andersson C, Gestrelius S (2001) Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative. J Clin Periodontol 28:181–188

    CAS  PubMed  Google Scholar 

  18. Maehara Y, Anai H, Tamada R, Sugimachi K (1987) The ATP assay is more sensitive than the succinate dehydrogenase inhibition test for predicting cell viability. Eur J Cancer Clin Oncol 23:273–276

    Google Scholar 

  19. Mellonig JT (1999) Enamel matrix derivative for periodontal reconstructive surgery: technique and clinical and histologic case report. Int J Periodontics Restorative Dent 19:8–19

    CAS  PubMed  Google Scholar 

  20. Murray E, Provvedini D, Curran D, Catherwood B, Sussman H, Manolagas S (1987) Characterization of a human osteoblastic osteosarcoma cell line (SAOS2) with high bone alkaline phosphatase activity. J Bone Miner Res 2:231–238

    CAS  PubMed  Google Scholar 

  21. Petty RD, Sutherland LA, Hunter EM, Cree IA (1995) Comparison of MTT and ATP-based assays for the measurement of viable cell number. J Biolumin Chemilumin 10:29–34

    CAS  PubMed  Google Scholar 

  22. Pontoriero R, Wennström J, Lindhe J (1999) The use of barrier membranes and enamel matrix proteins in the treatment of angular bone defects. A prospective controlled clinical study. J Clin Periodontol 26:833–840

    CAS  PubMed  Google Scholar 

  23. Rodan SB, Imai Y, Thiede MA, Wesolowski G, Thompson D, Bar-Shavit Z, Shull S, Mann K, Rodan GA (1987) Characterization of a human osteosarcoma cell line (SaOs2) with osteoblastic properties. Cancer Res 47:4961–4966

    CAS  PubMed  Google Scholar 

  24. Schwartz Z, Carnes DL Jr, Pulliam R, Lohmann CH, Sylvia VL, Liu Y, Dean DD, Cochran DL, Boyan BD (2000) Porcine fetal enamel matrix derivative stimulates proliferation but not differentiation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentiation of osteoblast-like MG63 cells, and increases proliferation and differentiation of normal human osteoblast NHOst cells. J Periodontol 71:1287–1296

    CAS  PubMed  Google Scholar 

  25. Schwarz F, Rothamel D, Sculean A, Georg T, Scherbaum W, Becker J (2003) Effects of an Er:YAG laser and the Vector ultrasonic system on the biocompatibility of titanium implants in cultures of human osteoblast-like cells. Clin Oral Implants Res 14:784–792

    Article  PubMed  Google Scholar 

  26. Sculean A, Donos N, Windisch P, Brecx M, Gera I, Reich E, Karring T (1999) Healing of human intrabony defects following treatment with enamel matrix proteins or guided tissue regeneration. J Periodontal Res 34:310–322

    CAS  PubMed  Google Scholar 

  27. Sculean A, Windisch P, Chiantella GC, Donos N, Brecx M, Reich E (2001) Treatment of intrabony defects with enamel matrix proteins and guided tissue regeneration. A prospective controlled clinical study. J Clin Periodontol 28:397–403

    Google Scholar 

  28. Sculean A, Windisch P, Keglevich T, Fabi B, Lundgren E, Lyngstadaas PS (2002) Presence of an enamel matrix protein derivative on human teeth following periodontal surgery. Clin Oral Invest 6:183–187

    Google Scholar 

  29. Silvestri M, Ricci G, Rasperini G, Sartori S, Cattaneo V (2000) Comparison of treatments of infrabony defects with enamel matrix derivative, guided tissue regeneration with a nonresorbable membrane and Widman modified flap. A pilot study. J Clin Periodontol 27:603–610

    Article  CAS  PubMed  Google Scholar 

  30. Slavkin HC (1976) Towards a cellular and molecular understanding of periodontics. Cementogenesis revisited. J Periodontol 47:249–255

    CAS  PubMed  Google Scholar 

  31. Van der Pauw MT, Van den Bos T, Everts V, Beertsen W (2000) Enamel matrix-derived protein stimulates attachment of periodontal ligament fibroblasts and enhances alkaline phosphatase activity and transforming growth factor beta1 release of periodontal ligament and gingival fibroblasts. J Periodontol 71:31–43

    PubMed  Google Scholar 

  32. Yoneda S, Itoh D, Kuroda S, Kondo H, Umezawa A, Ohya K, Ohyama T, Kasugai S (2003) The effects of enamel matrix derivative (EMD) on osteoblastic cells in culture and bone regeneration in a rat skull defect. J Periodontal Res 38:333–342

    Article  CAS  PubMed  Google Scholar 

  33. Yukna RA, Mellonig JT (2000) Histologic evaluation of periodontal healing in humans following regenerative therapy with enamel matrix derivative. A 10-case series. J Periodontol 71:752–759

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Oral Surgery, Heinrich Heine University, Westdeutsche Kieferklinik, Moorenstrasse 5, 40225, Düsseldorf, Germany

    Frank Schwarz, Daniel Rothamel, Monika Herten & Jürgen Becker

  2. Section of Periodontology, Department of Conservative Dentistry and Periodontology, Johannes Gutenberg University, 55101, Mainz, Germany

    Anton Sculean

  3. German Diabetes Center, Heinrich Heine University, Düsseldorf, Germany

    Werner Scherbaum

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  1. Frank Schwarz
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  2. Daniel Rothamel
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  4. Anton Sculean
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  5. Werner Scherbaum
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  6. Jürgen Becker
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Correspondence to Frank Schwarz.

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Schwarz, F., Rothamel, D., Herten, M. et al. Effect of enamel matrix protein derivative on the attachment, proliferation, and viability of human SaOs2 osteoblasts on titanium implants. Clin Oral Invest 8, 165–171 (2004). https://doi.org/10.1007/s00784-004-0259-2

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  • DOI: https://doi.org/10.1007/s00784-004-0259-2

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