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Potential dental pulp revascularization and odonto-/osteogenic capacity of a novel transplant combined with dental pulp stem cells and platelet-rich fibrin

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Abstract

Our aim is to investigate the cytobiological effects of autologous platelet-rich fibrin (PRF) on dental pulp stem cells (DPSCs) and to explore the ectopic and orthotopic possibilities of dental pulp revascularization and pulp-dentin complex regeneration along the root canal cavities of the tooth by using a novel tissue-engineered transplant composed of cell-sheet fragments of DPSCs and PRF granules. Canine DPSCs were isolated and characterized by assaying their colony-forming ability and by determining their cell surface markers and osteogenic/adipogenic differentiation potential. The biological effects of autologous PRF on DPSCs, including cell proliferation, alkaline phosphatase (ALP) activity and odonto-/osteogenic gene expression, were then investigated and quantified. A novel transplant consisting of cell-sheet fragments of DPSCs and PRF granules was adopted to regenerate pulp-dentin-like tissues in the root canal, both subcutaneously in nude mice and in the roots of canines. PRF promoted the proliferation of DPSCs in a dose- and time-dependent manner and induced the differentiation of DPSCs to odonto-/osteoblastic fates by increasing the expression of the Alp, Dspp, Dmp1 and Bsp genes. Transplantation of the DPSC/PRF construct led both to a favorable regeneration of homogeneous and compact pulp-like tissues with abundantly distributed blood capillaries and to the deposition of regenerated dentin along the intracanal walls at 8 weeks post-operation. Thus, the application of DPSC/PRF tissue constructs might serve as a potential therapy in regenerative endodontics for pulp revitalization or revascularization.

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References

  1. Almushayt A, Narayanan K, Zaki AE, George A (2006) Dentin matrix protein 1 induces cytodifferentiation of dental pulp stem cells into odontoblasts. Gene Ther 13:611–620

  2. Becerra P, Ricucci D, Loghin S, Gibbs JL, Lin LM (2014) Histologic study of a human immature permanent premolar with chronic apical abscess after revascularization/revitalization. J Endod 40:133–139

  3. Chadipiralla K, Yochim JM, Bahuleyan B, Huang CY, Garcia-Godoy F, Murray PE, Stelnicki EJ (2010) Osteogenic differentiation of stem cells derived from human periodontal ligaments and pulp of human exfoliated deciduous teeth. Cell Tissue Res 340:323–333

  4. Cordeiro MM, Dong Z, Kaneko T, Zhang Z, Miyazawa M, Shi S, Smith AJ, Nor JE (2008) Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth. J Endod 34:962–969

  5. da Silva LA, Nelson-Filho P, da Silva RA, Flores DS, Heilborn C, Johnson JD, Cohenca N (2010) Revascularization and periapical repair after endodontic treatment using apical negative pressure irrigation versus conventional irrigation plus triantibiotic intracanal dressing in dogs’ teeth with apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 109:779–787

  6. Dissanayaka WL, Zhu X, Zhang C, Jin L (2011) Characterization of dental pulp stem cells isolated from canine premolars. J Endod 37:1074–1080

  7. Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi J, Gogly B (2006) Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I. Technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101:e37–e44

  8. Dohan Ehrenfest DM, Rasmusson L, Albrektsson T (2009) Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends Biotechnol 27:158–167

  9. Dohan Ehrenfest DM, Doglioli P, de Peppo GM, Del Corso M, Charrier JB (2010) Choukroun’s platelet-rich fibrin (PRF) stimulates in vitro proliferation and differentiation of human oral bone mesenchymal stem cell in a dose-dependent way. Arch Oral Biol 55:185–194

  10. Fujita H, Shimizu K, Nagamori E (2009) Application of a cell sheet-polymer film complex with temperature sensitivity for increased mechanical strength and cell alignment capability. Biotechnol Bioeng 103:370–377

  11. Goncalves SB, Dong Z, Bramante CM, Holland GR, Smith AJ, Nor JE (2007) Tooth slice-based models for the study of human dental pulp angiogenesis. J Endod 33:811–814

  12. 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 U S A 97:13625–13630

  13. 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

  14. Gu K, Chang S, Ritchie HH, Clarkson BH, Rutherford RB (2000) Molecular cloning of a human dentin sialophosphoprotein gene. Eur J Oral Sci 108:35–42

  15. Gundberg CM (2000) Biochemical markers of bone formation. Clin Lab Med 20:489–501

  16. Hargreaves KM, Geisler T, Henry M, Wang Y (2008) Regeneration potential of the young permanent tooth: what does the future hold? Pediatr Dent 30:253–260

  17. He G, Dahl T, Veis A, George A (2003) Dentin matrix protein 1 initiates hydroxyapatite formation in vitro. Connect Tissue Res 44 (Suppl 1):240–245

  18. He L, Lin Y, Hu X, Zhang Y, Wu H (2009) A comparative study of platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) on the effect of proliferation and differentiation of rat osteoblasts in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108:707–713

  19. Huang GT, Garcia-Godoy F (2014) Missing concepts in de novo pulp regeneration. J Dent Res 93:717–724

  20. Huang GT, Shagramanova K, Chan SW (2006a) Formation of odontoblast-like cells from cultured human dental pulp cells on dentin in vitro. J Endod 32:1066–1073

  21. Huang GT, Sonoyama W, Chen J, Park SH (2006b) In vitro characterization of human dental pulp cells: various isolation methods and culturing environments. Cell Tissue Res 324:225–236

  22. Huang GT, Sonoyama W, Liu Y, Liu H, Wang S, Shi S (2008) The hidden treasure in apical papilla: the potential role in pulp/dentin regeneration and bioroot engineering. J Endod 34:645–651

  23. Huang FM, Yang SF, Zhao JH, Chang YC (2010a) Platelet-rich fibrin increases proliferation and differentiation of human dental pulp cells. J Endod 36:1628–1632

  24. Huang GT, Yamaza T, Shea LD, Djouad F, Kuhn NZ, Tuan RS, Shi S (2010b) Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng A 16:605–615

  25. Hung CN, Mar K, Chang HC, Chiang YL, Hu HY, Lai CC, Chu RM, Ma CM (2011) A comparison between adipose tissue and dental pulp as sources of MSCs for tooth regeneration. Biomaterials 32:6995–7005

  26. Iohara K, Nakashima M, Ito M, Ishikawa M, Nakasima A, Akamine A (2004) Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2. J Dent Res 83:590–595

  27. Iohara K, Imabayashi K, Ishizaka R, Watanabe A, Nabekura J, Ito M, Matsushita K, Nakamura H, Nakashima M (2011) Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1. Tissue Eng A 17:1911–1920

  28. Ishizaka R, Iohara K, Murakami M, Fukuta O, Nakashima M (2012) Regeneration of dental pulp following pulpectomy by fractionated stem/progenitor cells from bone marrow and adipose tissue. Biomaterials 33:2109–2118

  29. Ishizaka R, Hayashi Y, Iohara K, Sugiyama M, Murakami M, Yamamoto T, Fukuta O, Nakashima M (2013) Stimulation of angiogenesis, neurogenesis and regeneration by side population cells from dental pulp. Biomaterials 34:1888–1897

  30. Jiang HB, Tian WD, Liu LK, Xu Y (2008) In vitro odontoblast-like cell differentiation of cranial neural crest cells induced by fibroblast growth factor 8 and dentin non-collagen proteins. Cell Biol Int 32:671–678

  31. Kawashima N (2012) Characterisation of dental pulp stem cells: a new horizon for tissue regeneration? Arch Oral Biol 57:1439–1458

  32. Kelm JM, Fussenegger M (2010) Scaffold-free cell delivery for use in regenerative medicine. Adv Drug Deliv Rev 62:753–764

  33. Kim JY, Xin X, Moioli EK, Chung J, Lee CH, Chen M, Fu SY, Koch PD, Mao JJ (2010) Regeneration of dental-pulp-like tissue by chemotaxis-induced cell homing. Tissue Eng A 16:3023–3031

  34. Lee UL, Jeon SH, Park JY, Choung PH (2011) Effect of platelet-rich plasma on dental stem cells derived from human impacted third molars. Regen Med 6:67–79

  35. Li ZY, Chen L, Liu L, Lin YF, Li SW, Tian WD (2007) Odontogenic potential of bone marrow mesenchymal stem cells. J Oral Maxillofac Surg 65:494–500

  36. Lin LM, Ricucci D, Huang GT (2013) Regeneration of the dentine-pulp complex with revitalization/revascularization therapy: challenges and hopes. Int Endod J 47:713–724

  37. Liu H, Li W, Gao C, Kumagai Y, Blacher RW, DenBesten PK (2004) Dentonin, a fragment of MEPE, enhanced dental pulp stem cell proliferation. J Dent Res 83:496–499

  38. Mao JJ, Kim SG, Zhou J, Ye L, Cho S, Suzuki T, Fu SY, Yang R, Zhou X (2012) Regenerative endodontics: barriers and strategies for clinical translation. Dent Clin N Am 56:639–649

  39. Matsubara T, Suardita K, Ishii M, Sugiyama M, Igarashi A, Oda R, Nishimura M, Saito M, Nakagawa K, Yamanaka K, Miyazaki K, Shimizu M, Bhawal UK, Tsuji K, Nakamura K, Kato Y (2005) Alveolar bone marrow as a cell source for regenerative medicine: differences between alveolar and iliac bone marrow stromal cells. J Bone Miner Res 20:399–409

  40. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S (2003) SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 100:5807–5812

  41. Murray PE, Garcia-Godoy F, Hargreaves KM (2007) Regenerative endodontics: a review of current status and a call for action. J Endod 33:377–390

  42. Nakashima M, Huang GT (2013) Pulp and dentin regeneration. In: Huang GT, Thesleff I (eds) Stem cells in craniofacial development and regeneration. Wiley-Blackwell, Hoboken

  43. 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

  44. Nakashima M, Iohara K, Sugiyama M (2009) Human dental pulp stem cells with highly angiogenic and neurogenic potential for possible use in pulp regeneration. Cytokine Growth Factor Rev 20:435–440

  45. Narayanan K, Ramachandran A, Hao J, He G, Park KW, Cho M, George A (2003) Dual functional roles of dentin matrix protein 1. Implications in biomineralization and gene transcription by activation of intracellular Ca2+ store. J Biol Chem 278:17500–17508

  46. Ohazama A, Modino SA, Miletich I, Sharpe PT (2004) Stem-cell-based tissue engineering of murine teeth. J Dent Res 83:518–522

  47. 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

  48. Prescott RS, Alsanea R, Fayad MI, Johnson BR, Wenckus CS, Hao J, John AS, George A (2008) In vivo generation of dental pulp-like tissue by using dental pulp stem cells, a collagen scaffold, and dentin matrix protein 1 after subcutaneous transplantation in mice. J Endod 34:421–426

  49. Qin C, Brunn JC, Cadena E, Ridall A, Tsujigiwa H, Nagatsuka H, Nagai N, Butler WT (2002) The expression of dentin sialophosphoprotein gene in bone. J Dent Res 81:392–394

  50. Rosa V, Zhang Z, Grande RH, Nor JE (2013) Dental pulp tissue engineering in full-length human root canals. J Dent Res 92:970–975

  51. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364:149–155

  52. Shiba H, Fujita T, Doi N, Nakamura S, Nakanishi K, Takemoto T, Hino T, Noshiro M, Kawamoto T, Kurihara H, Kato Y (1998) Differential effects of various growth factors and cytokines on the syntheses of DNA, type I collagen, laminin, fibronectin, osteonectin/secreted protein, acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture. J Cell Physiol 174:194–205

  53. Shiba H, Nakanishi K, Rashid F, Mizuno N, Hino T, Ogawa T, Kurihara H (2003) Proliferative ability and alkaline phosphatase activity with in vivo cellular aging in human pulp cells. J Endod 29:9–11

  54. Shimizu E, Ricucci D, Albert J, Alobaid AS, Gibbs JL, Huang GT, Lin LM (2013) Clinical, radiographic, and histological observation of a human immature permanent tooth with chronic apical abscess after revitalization treatment. J Endod 39:1078–1083

  55. Sloan AJ, Smith AJ (2007) Stem cells and the dental pulp: potential roles in dentine regeneration and repair. Oral Dis 13:151–157

  56. Sonoyama W, Liu Y, Yamaza T, Tuan RS, Wang S, Shi S, Huang GT (2008) Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod 34:166–171

  57. Sumita Y, Honda MJ, Ohara T, Tsuchiya S, Sagara H, Kagami H, Ueda M (2006) Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering. Biomaterials 27:3238–3248

  58. Thibodeau B, Teixeira F, Yamauchi M, Caplan DJ, Trope M (2007) Pulp revascularization of immature dog teeth with apical periodontitis. J Endod 33:680–689

  59. Tsai C-H, Shen S-Y, Zhao J-H, Chang Y-C (2009) Platelet-rich fibrin modulates cel proliferation of human periodontally related cells in vitro. J Dent Sci 4:130–135

  60. Wang X, Thibodeau B, Trope M, Lin LM, Huang GT (2010) Histologic characterization of regenerated tissues in canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis. J Endod 36:56–63

  61. Wu G, Deng ZH, Fan XJ, Ma ZF, Sun YJ, Ma DD, Wu JJ, Shi JN, Jin Y (2009) Odontogenic potential of mesenchymal cells from hair follicle dermal papilla. Stem Cells Dev 18:583–589

  62. Yamauchi N, Nagaoka H, Yamauchi S, Teixeira FB, Miguez P, Yamauchi M (2011a) Immunohistological characterization of newly formed tissues after regenerative procedure in immature dog teeth. J Endod 37:1636–1641

  63. Yamauchi N, Yamauchi S, Nagaoka H, Duggan D, Zhong S, Lee SM, Teixeira FB, Yamauchi M (2011b) Tissue engineering strategies for immature teeth with apical periodontitis. J Endod 37:390–397

  64. Yang X, Walboomers XF, van den Beucken JJ, Bian Z, Fan M, Jansen JA (2009) Hard tissue formation of STRO-1-selected rat dental pulp stem cells in vivo. Tissue Eng A 15:367–375

  65. Yu J, Wang Y, Deng Z, Tang L, Li Y, Shi J, Jin Y (2007) Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells. Biol Cell 99:465–474

  66. Zhang W, Walboomers XF, van Kuppevelt TH, Daamen WF, Bian Z, Jansen JA (2006) The performance of human dental pulp stem cells on different three-dimensional scaffold materials. Biomaterials 27:5658–5668

  67. Zhao YH, Zhang M, Liu NX, Lv X, Zhang J, Chen FM, Chen YJ (2013) The combined use of cell sheet fragments of periodontal ligament stem cells and platelet-rich fibrin granules for avulsed tooth reimplantation. Biomaterials 34:5506–5520

  68. Zhu W, Zhu X, Huang GT, Cheung GS, Dissanayaka WL, Zhang C (2013) Regeneration of dental pulp tissue in immature teeth with apical periodontitis using platelet-rich plasma and dental pulp cells. Int Endod J 46:962–970

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Author information

Correspondence to Min Zhang.

Additional information

Yong-Jin Chen, Yin-Hua Zhao and Ya-Juan Zhao contributed equally to this work.

Animal experiments were undertaken after being approved by the Animal Experimental Ethical Inspection of FMMU (no. 12011).

This research was supported by the National Natural Science Foundation of China (nos. 81371188, 31170888).

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Chen, Y., Zhao, Y., Zhao, Y. et al. Potential dental pulp revascularization and odonto-/osteogenic capacity of a novel transplant combined with dental pulp stem cells and platelet-rich fibrin. Cell Tissue Res 361, 439–455 (2015). https://doi.org/10.1007/s00441-015-2125-8

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Keywords

  • Pulp revascularization
  • Dental pulp stem cells
  • Platelet-rich fibrin
  • Cell sheets
  • Regenerative dentistry