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Clinical Oral Investigations

, Volume 18, Issue 2, pp 443–451 | Cite as

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative

  • Richard J. Miron
  • Dieter D. Bosshardt
  • Anja C. Gemperli
  • Michel Dard
  • Daniel Buser
  • Reinhard Gruber
  • Anton SculeanEmail author
Original Article

Abstract

Objectives

Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials.

Aims

This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells.

Materials and methods

EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining.

Results

Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points.

Conclusion

The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings.

Clinical relevance

The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Keywords

Enamel matrix derivative (EMD) Emdogain Allograft Blood proteins Protein adsorption Bone grafting materials 

Notes

Acknowledgments

The authors gratefully acknowledge the Robert K. Schenk Laboratory of Oral Histology, School of Dental Medicine at the University of Bern, most notably Thuy Tran Nguyen and Monika Aeberhard for their considerable time and valuable insights into the project design. This work was funded by the Department of Periodontology at the University of Bern and Institut Straumann AG (Basel, Switzerland).

Conflict of interest

The authors report no conflicts of interest related to this study.

References

  1. 1.
    Rickert D, Slater JJ, Meijer HJ, Vissink A, Raghoebar GM (2012) Maxillary sinus lift with solely autogenous bone compared to a combination of autogenous bone and growth factors or (solely) bone substitutes. A systematic review. Int J Oral Maxillofac Surg 41(2):160–167. doi: 10.1016/j.ijom.2011.10.001 PubMedCrossRefGoogle Scholar
  2. 2.
    Kuhl S, Brochhausen C, Gotz H, Filippi A, Payer M, d’Hoedt B, Kreisler M (2012) The influence of bone substitute materials on the bone volume after maxillary sinus augmentation: a microcomputerized tomography study. Clin Oral Investig. doi: 10.1007/s00784-012-0732-2 Google Scholar
  3. 3.
    Huang HL, Hsu JT, Chen MY, Liu C, Chang CH, Li YF, Chen KT (2012) Microcomputed tomography analysis of particular autogenous bone graft in sinus augmentation at 5 months: differences on bone mineral density and 3D trabecular structure. Clin Oral Investig. doi: 10.1007/s00784-012-0725-1 Google Scholar
  4. 4.
    Horvath A, Stavropoulos A, Windisch P, Lukacs L, Gera I, Sculean A (2012) Histological evaluation of human intrabony periodontal defects treated with an unsintered nanocrystalline hydroxyapatite paste. Clin Oral Investig. doi: 10.1007/s00784-012-0739-8 Google Scholar
  5. 5.
    Pietruska M, Pietruski J, Nagy K, Brecx M, Arweiler NB, Sculean A (2012) Four-year results following treatment of intrabony periodontal defects with an enamel matrix derivative alone or combined with a biphasic calcium phosphate. Clin Oral Investig 16(4):1191–1197. doi: 10.1007/s00784-011-0611-2 PubMedCrossRefGoogle Scholar
  6. 6.
    van Hout WM, Mink van der Molen AB, Breugem CC, Koole R, Van Cann EM (2011) Reconstruction of the alveolar cleft: can growth factor-aided tissue engineering replace autologous bone grafting? A literature review and systematic review of results obtained with bone morphogenetic protein-2. Clin Oral Investig 15(3):297–303. doi: 10.1007/s00784-011-0547-6 PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Nauth A, Ristevski B, Li R, Schemitsch EH (2011) Growth factors and bone regeneration: how much bone can we expect? Injury 42(6):574–579. doi: 10.1016/j.injury.2011.03.034 PubMedCrossRefGoogle Scholar
  8. 8.
    Browaeys H, Bouvry P, De Bruyn H (2007) A literature review on biomaterials in sinus augmentation procedures. Clin Implant Dent Relat Res 9(3):166–177. doi: 10.1111/j.1708-8208.2007.00050.x PubMedCrossRefGoogle Scholar
  9. 9.
    Jensen T, Schou S, Stavropoulos A, Terheyden H, Holmstrup P (2012) Maxillary sinus floor augmentation with Bio-Oss or Bio-Oss mixed with autogenous bone as graft: a systematic review. Clin Oral Implants Res 23(3):263–273. doi: 10.1111/j.1600-0501.2011.02168.x PubMedCrossRefGoogle Scholar
  10. 10.
    Miron RJ, Zhang YF (2012) Osteoinduction: a review of old concepts with new standards. J Dent Res 91(8):736–744. doi: 10.1177/0022034511435260 PubMedCrossRefGoogle Scholar
  11. 11.
    Yuan H, Yang Z, Li Y, Zhang X, De Bruijn JD, De Groot K (1998) Osteoinduction by calcium phosphate biomaterials. J Mater Sci Mater Med 9(12):723–726PubMedCrossRefGoogle Scholar
  12. 12.
    Fellah BH, Gauthier O, Weiss P, Chappard D, Layrolle P (2008) Osteogenicity of biphasic calcium phosphate ceramics and bone autograft in a goat model. Biomaterials 29(9):1177–1188. doi: 10.1016/j.biomaterials.2007.11.034 PubMedCrossRefGoogle Scholar
  13. 13.
    Yuan H, Fernandes H, Habibovic P, de Boer J, Barradas AM, de Ruiter A, Walsh WR, van Blitterswijk CA, de Bruijn JD (2010) Osteoinductive ceramics as a synthetic alternative to autologous bone grafting. Proc Natl Acad Sci U S A 107(31):13614–13619. doi: 10.1073/pnas.1003600107 PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Sculean A, Alessandri R, Miron RJ, Salvi G, Bosshard DD (2011) Enamel matrix proteins and periodontal wound healing and regeneration. Clin Adv Periodontics 1:101–117CrossRefGoogle Scholar
  15. 15.
    Polimeni G, Koo KT, Qahash M, Xiropaidis AV, Albandar JM, Wikesjo UM (2004) Prognostic factors for alveolar regeneration: effect of tissue occlusion on alveolar bone regeneration with guided tissue regeneration. J Clin Periodontol 31(9):730–735. doi: 10.1111/j.1600-051X.2004.00543.x PubMedCrossRefGoogle Scholar
  16. 16.
    Siciliano VI, Andreuccetti G, Siciliano AI, Blasi A, Sculean A, Salvi GE (2011) Clinical outcomes after treatment of non-contained intrabony defects with enamel matrix derivative or guided tissue regeneration: a 12-month randomized controlled clinical trial. J Periodontol 82(1):62–71. doi: 10.1902/jop.2010.100144 PubMedCrossRefGoogle Scholar
  17. 17.
    Aspriello SD, Ferrante L, Rubini C, Piemontese M (2011) Comparative study of DFDBA in combination with enamel matrix derivative versus DFDBA alone for treatment of periodontal intrabony defects at 12 months post-surgery. Clin Oral Investig 15(2):225–232. doi: 10.1007/s00784-009-0369-y PubMedCrossRefGoogle Scholar
  18. 18.
    Hoidal MJ, Grimard BA, Mills MP, Schoolfield JD, Mellonig JT, Mealey BL (2008) Clinical evaluation of demineralized freeze-dried bone allograft with and without enamel matrix derivative for the treatment of periodontal osseous defects in humans. J Periodontol 79(12):2273–2280. doi: 10.1902/jop.2008.080259 PubMedCrossRefGoogle Scholar
  19. 19.
    Intini G, Andreana S, Buhite RJ, Bobek LA (2008) A comparative analysis of bone formation induced by human demineralized freeze-dried bone and enamel matrix derivative in rat calvaria critical-size bone defects. J Periodontol 79(7):1217–1224. doi: 10.1902/jop.2008.070435 PubMedCrossRefGoogle Scholar
  20. 20.
    Boyan BD, Weesner TC, Lohmann CH, Andreacchio D, Carnes DL, Dean DD, Cochran DL, Schwartz Z (2000) Porcine fetal enamel matrix derivative enhances bone formation induced by demineralized freeze dried bone allograft in vivo. J Periodontol 71(8):1278–1286. doi: 10.1902/jop.2000.71.8.1278 PubMedCrossRefGoogle Scholar
  21. 21.
    Gurinsky BS, Mills MP, Mellonig JT (2004) Clinical evaluation of demineralized freeze-dried bone allograft and enamel matrix derivative versus enamel matrix derivative alone for the treatment of periodontal osseous defects in humans. J Periodontol 75(10):1309–1318. doi: 10.1902/jop.2004.75.10.1309 PubMedCrossRefGoogle Scholar
  22. 22.
    Rosen PS, Reynolds MA (2002) A retrospective case series comparing the use of demineralized freeze-dried bone allograft and freeze-dried bone allograft combined with enamel matrix derivative for the treatment of advanced osseous lesions. J Periodontol 73(8):942–949. doi: 10.1902/jop.2002.73.8.942 PubMedCrossRefGoogle Scholar
  23. 23.
    Cochran DL, Jones A, Heijl L, Mellonig JT, Schoolfield J, King GN (2003) Periodontal regeneration with a combination of enamel matrix proteins and autogenous bone grafting. J Periodontol 74(9):1269–1281. doi: 10.1902/jop.2003.74.9.1269 PubMedCrossRefGoogle Scholar
  24. 24.
    Yamamoto S, Masuda H, Shibukawa Y, Yamada S (2007) Combination of bovine-derived xenografts and enamel matrix derivative in the treatment of intrabony periodontal defects in dogs. Int J Periodontics Restorative Dent 27(5):471–479PubMedGoogle Scholar
  25. 25.
    Velasquez-Plata D, Scheyer ET, Mellonig JT (2002) Clinical comparison of an enamel matrix derivative used alone or in combination with a bovine-derived xenograft for the treatment of periodontal osseous defects in humans. J Periodontol 73(4):433–440. doi: 10.1902/jop.2002.73.4.433 PubMedCrossRefGoogle Scholar
  26. 26.
    Zucchelli G, Amore C, Montebugnoli L, De Sanctis M (2003) Enamel matrix proteins and bovine porous bone mineral in the treatment of intrabony defects: a comparative controlled clinical trial. J Periodontol 74(12):1725–1735. doi: 10.1902/jop.2003.74.12.1725 PubMedCrossRefGoogle Scholar
  27. 27.
    Guida L, Annunziata M, Belardo S, Farina R, Scabbia A, Trombelli L (2007) Effect of autogenous cortical bone particulate in conjunction with enamel matrix derivative in the treatment of periodontal intraosseous defects. J Periodontol 78(2):231–238. doi: 10.1902/jop.2007.060142 PubMedCrossRefGoogle Scholar
  28. 28.
    Yilmaz S, Cakar G, Yildirim B, Sculean A (2010) Healing of two and three wall intrabony periodontal defects following treatment with an enamel matrix derivative combined with autogenous bone. J Clin Periodontol 37(6):544–550. doi: 10.1111/j.1600-051X.2010.01567.x PubMedCrossRefGoogle Scholar
  29. 29.
    Sculean A, Windisch P, Keglevich T, Gera I (2005) Clinical and histologic evaluation of an enamel matrix protein derivative combined with a bioactive glass for the treatment of intrabony periodontal defects in humans. Int J Periodontics Restorative Dent 25(2):139–147PubMedGoogle Scholar
  30. 30.
    Sculean A, Windisch P, Szendroi-Kiss D, Horvath A, Rosta P, Becker J, Gera I, Schwarz F (2008) Clinical and histologic evaluation of an enamel matrix derivative combined with a biphasic calcium phosphate for the treatment of human intrabony periodontal defects. J Periodontol 79(10):1991–1999. doi: 10.1902/jop.2008.080009 PubMedCrossRefGoogle Scholar
  31. 31.
    Miron RJ, Bosshardt D, Hedbom E, Zhang Y, Haenni B, Buser D, Sculean A (2012) Adsorption of enamel matrix proteins to a bovine derived bone grafting material and its regulation of cell adhesion, proliferation and differentiation. J Periodontol. doi: 10.1902/jop.2011.110480 Google Scholar
  32. 32.
    Miron RJ, Gruber R, Hedbom E, Saulacic N, Zhang Y, Sculean A, Bosshardt DD, Buser D (2012) Impact of bone harvesting techniques on cell viability and the release of growth factors of autografts. Clin Implant Dent Relat Res. doi: 10.1111/j.1708-8208.2012.00440.x PubMedGoogle Scholar
  33. 33.
    Miron RJ, Oates CJ, Molenberg A, Dard M, Hamilton DW (2010) The effect of enamel matrix proteins on the spreading, proliferation and differentiation of osteoblasts cultured on titanium surfaces. Biomaterials 31(3):449–460. doi: 10.1016/j.biomaterials.2009.09.075 PubMedCrossRefGoogle Scholar
  34. 34.
    Miron RJ, Hedbom E, Saulacic N, Zhang Y, Sculean A, Bosshardt DD, Buser D (2011) Osteogenic potential of autogenous bone grafts harvested with four different surgical techniques. J Dent Res 90(12):1428–1433. doi: 10.1177/0022034511422718 PubMedCrossRefGoogle Scholar
  35. 35.
    Yuan H, van Blitterswijk CA, de Groot K, de Bruijn JD (2006) Cross-species comparison of ectopic bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) scaffolds. Tissue Eng 12(6):1607–1615. doi: 10.1089/ten.2006.12.1607 PubMedCrossRefGoogle Scholar
  36. 36.
    Bosshardt DD (2008) Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels. J Clin Periodontol 35(8 Suppl):87–105. doi: 10.1111/j.1600-051X.2008.01264.x PubMedCrossRefGoogle Scholar
  37. 37.
    Miron RJ, Hedbom E, Ruggiero S, Bosshardt DD, Zhang Y, Mauth C, Gemperli AC, Iizuka T, Buser D, Sculean A (2011) Premature osteoblast clustering by enamel matrix proteins induces osteoblast differentiation through up-regulation of connexin 43 and N-cadherin. PLoS One 6(8):e23375. doi: 10.1371/journal.pone.0023375 PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    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(9 Pt 2):678–684PubMedCrossRefGoogle Scholar
  39. 39.
    Suzuki S, Nagano T, Yamakoshi Y, Gomi K, Arai T, Fukae M, Katagiri T, Oida S (2005) Enamel matrix derivative gel stimulates signal transduction of BMP and TGF-{beta}. J Dent Res 84(6):510–514PubMedCrossRefGoogle Scholar
  40. 40.
    Dean DD, Lohmann CH, Sylvia VL, Cochran DL, Liu Y, Boyan BD, Schwartz Z (2002) Effect of porcine fetal enamel matrix derivative on chondrocyte proliferation, differentiation, and local factor production is dependent on cell maturation state. Cells Tissues Organs 171(2–3):117–127PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Richard J. Miron
    • 1
    • 2
  • Dieter D. Bosshardt
    • 1
    • 2
  • Anja C. Gemperli
    • 3
  • Michel Dard
    • 3
  • Daniel Buser
    • 2
  • Reinhard Gruber
    • 1
    • 2
  • Anton Sculean
    • 1
    Email author
  1. 1.Department of Periodontology, School of Dental MedicineUniversity of BernBernSwitzerland
  2. 2.Department of Oral Surgery and Stomatology, School of Dental MedicineUniversity of BernBernSwitzerland
  3. 3.Institute Straumann AGBaselSwitzerland

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