title: Engineering three-dimensional constructs of the periodontal ligament in hyaluronan-gelatin hydrogel films and a mechanically active environment.
abstract: OBJECTIVE
  Periodontal ligament (PDL) cells in stationary two-dimensional culture systems are in a double default state. Our aim therefore was to engineer and characterize three-dimensional constructs, by seeding PDL cells into hyaluronan-gelatin hydrogel films (80-100 μm) in a format capable of being mechanically deformed.
  
  
  METHODS
  Human PDL constructs were cultured with and without connective tissue growth factor (CTGF) and fibroblast growth factor (FGF)-2 in (i) stationary cultures, and (ii) mechanically active cultures subjected to cyclic strains of 12% at 0.2 Hz each min, 6 h/d, in a Flexercell FX-4000 Strain Unit. The following parameters were measured: cell number and viability by laser scanning confocal microscopy; cell proliferation with the MTS assay; the expression of a panel of 18 genes using real-time RT-PCR; matrix metalloproteinases (MMPs) 1-3, TIMP-1, CTGF and FGF-2 protein levels in supernatants from mechanically activated cultures with Enzyme-linked immunosorbent assays. Constructs from stationary cultures were also examined by scanning electron microscopy and immunostained for actin and vinculin.
  
  
  RESULTS
  Although initially randomly distributed, the cells became organized into a bilayer by day 7; apoptotic cells remained constant at approximately 5% of the total. CTGF/FGF-2 stimulated cell proliferation in stationary cultures, but relative quantity values suggested modest effects on gene expression. Two transcription factors (RUNX2 and PPARG), two collagens (COL1A1, COL3A1), four MMPs (MMP-1-3, TIMP-1), TGFB1, RANKL, OPG and P4HB were detected by gel electrophoresis and Ct values < 35. In mechanically active cultures, with the exception of P4HB, TGFB1 and RANKL, each was upregulated at some point in the time scale, as was the synthesis of MMPs and TIMP-1. SOX9, MYOD, SP7, BMP2, BGLAP or COL2A1 were not detected in either stationary or mechanically active cultures.
  
  
  CONCLUSIONS
  Three-dimensional tissue constructs provide additional complexity to monolayer culture systems, and suggest some of the assumptions regarding cell growth, differentiation and matrix turnover based on two-dimensional cultures may not apply to cells in three-dimensional matrices. Primarily developed as a transitional in vitro model for studying cell-cell and cell-matrix interactions in tooth support, the system is also suitable for investigating the pathogenesis of periodontal diseases, and importantly from the clinical point of view, in a mechanically active environment.
authors.affiliation: Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, Singapore, 119083, Singapore.
authors.forename: A
authors.initials: A
authors.lastname: Saminathan
authors.forename: K J
authors.initials: KJ
authors.lastname: Vinoth
authors.forename: H H
authors.initials: HH
authors.lastname: Low
authors.forename: T
authors.initials: T
authors.lastname: Cao
authors.forename: M C
authors.initials: MC
authors.lastname: Meikle
mesh_terms: D000199:Actins / Q000032:analysis; D001696:Biomechanical Phenomena; D002452:Cell Count; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002470:Cell Survival / Q000502:physiology; D055513:Connective Tissue Growth Factor / Q000032:analysis / Q000494:pharmacology; D003470:Culture Media; D016222:Fibroblast Growth Factor 2 / Q000032:analysis / Q000494:pharmacology; D005780:Gelatin / Q000737:chemistry*; D020869:Gene Expression Profiling; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry*; D020100:Hydrogels / Q000737:chemistry*; D020781:Matrix Metalloproteinase 1 / Q000032:analysis; D020778:Matrix Metalloproteinase 2 / Q000032:analysis; D019278:Matrix Metalloproteinase 3 / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D010513:Periodontal Ligament / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D018583:Pliability; D013314:Stress, Mechanical; D023822:Tissue Engineering / Q000379:methods*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis; D054457:Tissue Scaffolds / Q000737:chemistry*; D016596:Vinculin / Q000032:analysis
chemical_list: D000199:Actins; C526317:CCN2 protein, human; D003470:Culture Media; D020100:Hydrogels; D008565:Membrane Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; C488955:VCL protein, human; D016222:Fibroblast Growth Factor 2; D016596:Vinculin; D055513:Connective Tissue Growth Factor; D005780:Gelatin; D006820:Hyaluronic Acid; C511768:MMP3 protein, human; D019278:Matrix Metalloproteinase 3; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1
keywords: growth factors; in vitro model; matrix metalloproteinase; periodontal ligament; real-time RT-PCR