title: Expression of osteoblastic phenotype in periodontal ligament fibroblasts cultured in three-dimensional collagen gel.
abstract: OBJECTIVE
  To investigate the influence of a three-dimensional cell culture model on the expression of osteoblastic phenotype in human periodontal ligament fibroblast (hPDLF) cultures.
  
  
  METHODS
  hPDLF were seeded on bi-dimensional (2D) and three-dimensional (3D) collagen type I (experimental groups) and and on a plastic coverslip (control) for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity were performed. Also, cell morphology and immunolabeling for alkaline phosphatase (ALP) and osteopontin (OPN) were assessed by epifluorescence and confocal microscopy. The expression of osteogenic markers, including alkaline phosphatase, osteopontin, osteocalcin (OC), collagen I (COL I) and runt-related transcription factor 2 (RUNX2), were analyzed using real-time polymerase chain reaction (RT-PCR). Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay.
  
  
  RESULTS
  Experimental cultures produced an increase in cell proliferation. Immunolabeling for OPN and ALP in hPDLF were increased and ALP activity was inhibited by three-dimensional conditions. OPN and RUNX2 gene expression was significantly higher on 3D culture when compared with control surface. Moreover, ALP and COL I gene expression were significantly higher in three-dimensional collagen than in 2D cultures at 7 days. However, at 14 days, 3D cultures exhibited ALP and COL I gene expression significantly lower than the control, and the COL I gene expression was also significantly lower in 3D than in 2D cultures. Significant calcium mineralization was detected and quantified by alizarin red assay, and calcified nodule formation was not affected by tridimensionality.
  
  
  CONCLUSIONS
  This study suggests that the 3D cultures are able to support hPDLF proliferation and favor the differentiation and mineralized matrix formation, which may be a potential periodontal regenerative therapy.
authors.affiliation: Department of Oral Surgery and Periodontology, Ribeirão Preto School of Dentistry, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Luciana Bastos
authors.initials: LB
authors.lastname: Alves
authors.affiliation: Department of Oral Surgery and Periodontology, Ribeirão Preto School of Dentistry, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Viviane Casagrande
authors.initials: VC
authors.lastname: Mariguela
authors.affiliation: Department of Oral Surgery and Periodontology, Ribeirão Preto School of Dentistry, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Márcio Fernando de Moraes
authors.initials: MF
authors.lastname: Grisi
authors.affiliation: Department of Oral Surgery and Periodontology, Ribeirão Preto School of Dentistry, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Sérgio Luiz Scaombatti de
authors.initials: SL
authors.lastname: Souza
authors.affiliation: Department of Oral Surgery and Periodontology, Ribeirão Preto School of Dentistry, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Arthur Belém
authors.initials: AB
authors.lastname: Novaes Junior
authors.affiliation: Department of Oral Surgery and Periodontology, Ribeirão Preto School of Dentistry, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Mário
authors.initials: M
authors.lastname: Taba  Junior
authors.affiliation: Department of Morphology, Stomatology and Physiology, Ribeirão Preto School of Dentistry, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Paulo Tambasco de
authors.initials: PT
authors.lastname: Oliveira
authors.affiliation: Department of Morphology, Stomatology and Physiology, Ribeirão Preto School of Dentistry, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Daniela Bazan
authors.initials: DB
authors.lastname: Palioto
mesh_terms: D000469:Alkaline Phosphatase / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D024042:Collagen Type I / Q000494:pharmacology*; D050659:Core Binding Factor Alpha 1 Subunit / Q000502:physiology; D005347:Fibroblasts / Q000502:physiology*; D015870:Gene Expression; D006801:Humans; D010006:Osteoblasts / Q000502:physiology*; D015675:Osteocalcin / Q000502:physiology; D053495:Osteopontin / Q000502:physiology; D010513:Periodontal Ligament / Q000166:cytology*; D010641:Phenotype; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D018709:Statistics, Nonparametric; D013997:Time Factors
chemical_list: D024042:Collagen Type I; D050659:Core Binding Factor Alpha 1 Subunit; D015675:Osteocalcin; D053495:Osteopontin; D000469:Alkaline Phosphatase