title: EDTA soluble chemical components and the conditioned medium from mobilized dental pulp stem cells contain an inductive microenvironment, promoting cell proliferation, migration, and odontoblastic differentiation.
abstract: BACKGROUND
  The critical challenge in tissue engineering is to establish an optimal combination of stem cells, signaling morphogenetic molecules, and extracellular matrix scaffold/microenvironment. The extracellular matrix components of teeth may be reconstituted as an inductive microenvironment in an ectopic tooth transplantation bioassay. Thus, the isolation and identification of the chemical components of the inductive microenvironment in pulp/dentin regeneration will accelerate progress towards the goal of tissue engineering of the tooth.
  
  
  METHODS
  The teeth demineralized in 0.6 M hydrochloric acid were sequentially extracted by 4.0 M guanidine hydrochloride (GdnHCl), pH 7.4, and 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.4. The extracted teeth were transplanted into an ectopic site in severe combined immunodeficiency (SCID) mice with mobilized dental pulp stem cells (MDPSCs). The unextracted tooth served as a positive control. Furthermore, the soluble components for the inductive microenvironment, the GdnHCl extracts, or the EDTA extracts together with or without MDPSC conditioned medium (CM) were reconstituted systematically with autoclaved teeth in which the chemical components were completely inactivated and only the physical microenvironment was preserved. Their pulp/dentin regenerative potential and angiogenic potential were compared 28 days after ectopic tooth transplantation by histomorphometry and real-time RT-PCR analysis.
  
  
  RESULTS
  Expression of an odontoblastic marker, enamelysin, and a pulp marker, thyrotropin-releasing hormone degrading enzyme (TRH-DE), was lower, and expression of a periodontal cell marker, anti-asporin/periodontal ligament-associated protein 1 (PLAP-1), was higher in the transplant of the EDTA-extracted teeth compared with the GdnHCl-extracted teeth. The autoclaved teeth reconstituted with the GdnHCl extracts or the EDTA extracts have weak regenerative potential and minimal angiogenic potential, and the CM significantly increased this potential. Combinatorial effects of the EDTA extracts and the CM on pulp/dentin regeneration were demonstrated in vivo, consistent with their in-vitro effects on enhanced proliferation, migration, and odontoblastic differentiation.
  
  
  CONCLUSIONS
  The EDTA-extracted teeth demonstrated significantly lower pulp/dentin regenerative potential compared with the GdnHCl-extracted teeth. The EDTA soluble chemical components when reconstituted with the physical structure of autoclaved teeth serve as an inductive microenvironment for pulp/dentin regeneration, promoting cell proliferation, migration, and odontoblastic differentiation.
authors.affiliation: Department of Stem Cell Biology and Regenerative Medicine, National Center for Geriatrics and Gerontology, Research Institute, 7-430 Morioka, Obu, Aichi, 474-8511, Japan.|Department of Gerontology, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, 464-8651, Japan.|Department of Oral Implantology, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, 464-8651, Japan.
authors.forename: Rei
authors.initials: R
authors.lastname: Kawamura
authors.affiliation: Department of Stem Cell Biology and Regenerative Medicine, National Center for Geriatrics and Gerontology, Research Institute, 7-430 Morioka, Obu, Aichi, 474-8511, Japan.|Department of Pediatric Dentistry, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, 464-8651, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Hayashi
authors.affiliation: Department of Gerontology, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, 464-8651, Japan.|Department of Oral Implantology, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi, 464-8651, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Murakami
authors.affiliation: Department of Stem Cell Biology and Regenerative Medicine, National Center for Geriatrics and Gerontology, Research Institute, 7-430 Morioka, Obu, Aichi, 474-8511, Japan. misako@ncgg.go.jp.
authors.forename: Misako
authors.initials: M
authors.lastname: Nakashima
mesh_terms: D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001641:Bicuspid / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D060833:Cellular Microenvironment; D017077:Culture Media, Conditioned / Q000302:isolation & purification / Q000494:pharmacology*; D003782:Dental Pulp / Q000166:cytology / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D053519:Matrix Metalloproteinase 20 / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D009804:Odontoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013552:Swine; D023822:Tissue Engineering; D014183:Transplantation, Heterologous; D000099311:Dentin Sialophosphoprotein
chemical_list: D000626:Aminopeptidases; D015415:Biomarkers; D017077:Culture Media, Conditioned; D016326:Extracellular Matrix Proteins; D053519:Matrix Metalloproteinase 20; D010750:Phosphoproteins; D011761:Pyrrolidonecarboxylic Acid; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein; C023166:pyroglutamyl-peptidase II
keywords: Angiogenesis; Cell attachment; Chemical microenvironment; Dental pulp mesenchymal stem cells; Migration; Odontoblastic differentiation; Pulp/dentin regeneration