title: Coculture of dental pulp stem cells with endothelial cells enhances osteo-/odontogenic and angiogenic potential in vitro.
abstract: BACKGROUND
  Dental pulp stem cells (DPSCs) have received much attention as a promising population of stem cells in regenerative endodontics. Securing a good blood supply during regeneration is a challenging task because of the constricted apical canal opening, which allows only a limited blood supply. The aim of this study was to investigate any potential synergistic effects of dental pulp stem cells and endothelial cells (ECs) on osteo-/odontogenic and angiogenic differentiation in vitro.
  
  
  METHODS
  Different ratios of DPSCs and ECs were cultured in direct contact using optimized medium for coculture. The 70% confluent cocultures were incubated in the osteo-/odontogenic differentiation medium for up to 3 weeks. Alkaline phosphatase (ALP) activity, the expression levels of ALP, bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) genes, and alizarin red staining for mineralization at different time points were analyzed. The tubular network formation on Matrigel and the gene expression levels of CD117, VEGF, CD34, and Flk-1 were used as assays to analyze angiogenesis.
  
  
  RESULTS
  The quantification of ALP in DPSC:EC cocultures revealed a greater ALP activity compared with DPSC-alone cultures. At all the time points, 1:1 cultures showed a significantly greater ALP activity than that of DPSC-alone cultures. Alizarin red staining and quantification revealed a much greater amount of calcification in the 1:1 and 1:5 cocultures compared with other cultures (P < .01). The expression levels of ALP, BSP, and DSPP genes further confirmed the greater osteo-/odontogenic differentiation in cocultures compared with those of DPSC-alone cultures. Matrigel assay showed that the addition of DPSCs stabilized preexisting vessel-like structures formed by ECs and increased the longevity of them.
  
  
  CONCLUSIONS
  Direct coculture of DPSCs and ECs enhances the in vitro differentiation toward osteo-/odontogenic and angiogenic phenotypes.
authors.affiliation: Department of Endodontics, Comprehensive Dental Care, The University of Hong Kong, Hong Kong SAR, China.
authors.forename: Waruna Lakmal
authors.initials: WL
authors.lastname: Dissanayaka
authors.forename: Xuan
authors.initials: X
authors.lastname: Zhan
authors.forename: Chengfei
authors.initials: C
authors.lastname: Zhang
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Hargreaves
authors.forename: Lijian
authors.initials: L
authors.lastname: Jin
authors.forename: Edith H Y
authors.initials: EH
authors.lastname: Tong
mesh_terms: D000293:Adolescent; D000328:Adult; D000469:Alkaline Phosphatase / Q000032:analysis; D000880:Anthraquinones; D018952:Antigens, CD34 / Q000032:analysis; D002113:Calcification, Physiologic / Q000502:physiology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D018920:Coculture Techniques; D004396:Coloring Agents; D003470:Culture Media; D003782:Dental Pulp / Q000166:cytology*; D042783:Endothelial Cells / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology*; D016326:Extracellular Matrix Proteins / Q000032:analysis; D006801:Humans; D058574:Integrin-Binding Sialoprotein / Q000032:analysis; D055806:Microvessels / Q000254:growth & development; D018919:Neovascularization, Physiologic / Q000502:physiology*; D009805:Odontogenesis / Q000502:physiology*; D010012:Osteogenesis / Q000502:physiology*; D010750:Phosphoproteins / Q000032:analysis; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis; D012795:Sialoglycoproteins / Q000032:analysis; D013234:Stem Cells / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000032:analysis; D055815:Young Adult; D000099311:Dentin Sialophosphoprotein
chemical_list: D000469:Alkaline Phosphatase; D000880:Anthraquinones; D018952:Antigens, CD34; D004396:Coloring Agents; D003470:Culture Media; D016326:Extracellular Matrix Proteins; D058574:Integrin-Binding Sialoprotein; D010750:Phosphoproteins; D019009:Proto-Oncogene Proteins c-kit; D012795:Sialoglycoproteins; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D000099311:Dentin Sialophosphoprotein; C467484:VEGFA protein, human; C010078:alizarin