title: Real-time quantitative polymerase chain reaction and flow cytometric analyses of cell adhesion molecules expressed in human cell-multilayered periosteal sheets in vitro.
abstract: OBJECTIVE
  Cultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell-extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms of periosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, and three-dimensional hybrid mock-ups of cells dispersed onto collagen sponges.
  
  
  METHODS
  Periosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively.
  
  
  RESULTS
  Real-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (β1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of β1 integrin was substantially downregulated in the stem cell medium-expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets.
  
  
  CONCLUSIONS
  Integrin α1β1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets.
authors.affiliation: Division of Oral Bioengineering, Institute of Medicine and Dentistry, Niigata University, Niigata, Japan. Electronic address: kawase@dent.niigatau.ac.jp.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Kawase
authors.affiliation: Division of Oral Bioengineering, Institute of Medicine and Dentistry, Niigata University, Niigata, Japan; Division of Oral and Maxillofacial Surgery, Institute of Medicine and Dentistry, Niigata University, Niigata, Japan.
authors.forename: Kohya
authors.initials: K
authors.lastname: Uematsu
authors.affiliation: Division of Oral Bioengineering, Institute of Medicine and Dentistry, Niigata University, Niigata, Japan; Division of Periodontology, Institute of Medicine and Dentistry, Niigata University, Niigata, Japan.
authors.forename: Mana
authors.initials: M
authors.lastname: Kamiya
authors.affiliation: Division of Oral and Maxillofacial Surgery, Institute of Medicine and Dentistry, Niigata University, Niigata, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Nagata
authors.affiliation: Division of Periodontology, Institute of Medicine and Dentistry, Niigata University, Niigata, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Okuda
authors.affiliation: Midwest Biomedical Research Foundation, Research Service, Department of Veterans Affairs Medical Center, Kansas City, Missouri, USA.
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Burns
authors.affiliation: Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, Niigata, Japan.
authors.forename: Koh
authors.initials: K
authors.lastname: Nakata
authors.affiliation: Division of Periodontology, Institute of Medicine and Dentistry, Niigata University, Niigata, Japan.
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Yoshie
mesh_terms: D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D039222:Integrin alpha1beta1 / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D023822:Tissue Engineering
chemical_list: D015815:Cell Adhesion Molecules; D005353:Fibronectins; D018960:Hyaluronan Receptors; D039222:Integrin alpha1beta1
keywords: bone tissue engineering; cell adhesion; extracellular matrix; integrin; periosteal sheet