title: Osteoblast differentiation is enhanced in rotary cell culture simulated microgravity environments.
abstract: OBJECTIVE
  As the aging population increases, more people will become reliant on regenerative dental medicine for implant therapy. The objective of this study was to test the hypothesis that 3D rotary cell culture (RCC) environments created by simulated microgravity would enhance osteogenic gene expression using integrin mediated pathways.
  
  
  METHODS
  Human embryonic palatal mesenchymal (HEPM, ATCC 1486) pre-osteoblasts were cultured in either RCC to create 3D environments or in 2D monolayers for 72 hours. Gross phenotypic analysis was performed using Alizarin Red S staining for calcium and microscopy. Real-time PCR analysis was used to detect differences in osteoblast gene expression. Aggregates developed in 3D RCC environments were treated with or without antibody to the collagen-I integrin receptor alpha2beta1 to determine whether this molecular pathway might contribute to the development of a mineralized matrix.
  
  
  RESULTS
  Microscopic analysis demonstrated that RCC environments promoted 3D aggregate formation by 72 hours without any scaffold. The mass appeared osseous-like with a white, shiny, translucent surface. The center was amorphous with areas of vacuolization, tubule-like structures, and fibrous-like extensions. Real-time PCR data showed that 3D environments enhanced osteogenic gene expression as compared with 2D monolayer culturing conditions. At 72 hours, changes in levels of osteogenic gene expression were noted. Cbfa1, a necessary transcription factor for osteoblast differentiation, was expressed 33% higher (p= 0.26); Collagen 1, 69% higher (p= 0.05); Osterix, 49% higher (p= 0.001); and BSPII, 54% higher (p= 0.001) than osteoblasts cultured for 72 hours in standard 2D monolayer conditions. When cultured in the presence of collagen alpha2beta1 integrin receptor antibody, 3D aggregates had decreased levels of mineralization as compared with non-treated aggregates.
  
  
  CONCLUSIONS
  RCC enhances osteoblast differentiation using integrin mediated pathways.
authors.affiliation: Department of Prosthodontics, College of Dentistry, University of Iowa, Iowa City, IA, USA.
authors.forename: Y Joon
authors.initials: YJ
authors.lastname: Ko
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Zaharias
authors.forename: Denise A
authors.initials: DA
authors.lastname: Seabold
authors.forename: John
authors.initials: J
authors.lastname: Lafoon
authors.forename: Galen B
authors.initials: GB
authors.lastname: Schneider
mesh_terms: D000880:Anthraquinones; D002113:Calcification, Physiologic / Q000235:genetics; D002118:Calcium / Q000032:analysis; D002448:Cell Adhesion / Q000502:physiology; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000032:analysis; D004396:Coloring Agents; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis; D006801:Humans; D038982:Integrin alpha2beta1 / Q000032:analysis; D058574:Integrin-Binding Sialoprotein; D008648:Mesoderm / Q000166:cytology; D010006:Osteoblasts / Q000502:physiology*; D010012:Osteogenesis / Q000502:physiology*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012399:Rotation; D012795:Sialoglycoproteins / Q000032:analysis; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000032:analysis; D018474:Weightlessness Simulation
chemical_list: D000880:Anthraquinones; D024042:Collagen Type I; D004396:Coloring Agents; D050659:Core Binding Factor Alpha 1 Subunit; C546871:IBSP protein, human; D038982:Integrin alpha2beta1; D058574:Integrin-Binding Sialoprotein; D012795:Sialoglycoproteins; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors; C004468:Alizarin Red S; D002118:Calcium