title: Effects of osteogenic differentiation inducers on in vitro expanded adult mesenchymal stromal cells.
abstract: OBJECTIVE
  For bone regeneration therapy using stem cells, well-defined ex vivo protocols to expand mesenchymal stromal cells (MSC), as well as assays to show their potential differentiation into the osteogenic lineage, are needed. Aim of this study was to analyze the role of the biochemical osteogenic inducers, i.e. ascorbic acid, dexamethasone, and ß-glycerophosphate, employed in the current protocols for osteogenic differentiation of MSC in vitro, to address the requirements for reliable differentiation systems.
  
  
  METHODS
  MSC were isolated from the bone marrow of donors (46-73 years of age) undergoing total hip replacement, and expanded in vitro. At confluence, MSC were cultured under four different conditions: α-MEM plus serum (basal medium or C1), basal medium plus ascorbate (C2), basal medium plus ascorbate and dexamethasone (C3), or basal medium plus ascorbate, dexamethasone and ß-glycerophosphate (C4). Morphology, proliferation, mineralization, alkaline phosphatase, collagen and expression of bone-related genes of MSC under the different media were analyzed at fixed time points.
  
  
  RESULTS
  MSC proliferation and the number of colony forming units were increased by ascorbic acid, whereas dexamethasone enhanced the proportion of ALP-positive CFU and was critical for mineral deposition. Runx-2 and type I collagen gene expression decreased along with additive-induced MSC differentiation, i.e. from C1 to C4, while ALP and osteocalcin were differently regulated.
  
  
  CONCLUSIONS
  Our findings support the role of different inducers on the sequential stages of MSC expansion and osteogenic differentiation in vitro, suggesting the addition of DEX following proliferation to ensure mineralization, as an index of in vivo osteogenic potency of human mesenchymal cells.
authors.affiliation: Unit of Orthopedic Pathophysiology and Regenerative Medicine, Rizzoli Orthopedic Institute, Bologna, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Fiorentini
authors.forename: Donatella
authors.initials: D
authors.lastname: Granchi
authors.forename: Elisa
authors.initials: E
authors.lastname: Leonardi
authors.forename: Nicola
authors.initials: N
authors.lastname: Baldini
authors.forename: Gabriela
authors.initials: G
authors.lastname: Ciapetti
mesh_terms: D000368:Aged; D000469:Alkaline Phosphatase / Q000378:metabolism; D001205:Ascorbic Acid / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D005260:Female; D005994:Glycerophosphates / Q000494:pharmacology*; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D008875:Middle Aged; D010012:Osteogenesis / Q000187:drug effects* / Q000502:physiology
chemical_list: D024042:Collagen Type I; D005994:Glycerophosphates; D003907:Dexamethasone; D000469:Alkaline Phosphatase; D001205:Ascorbic Acid; C031463:beta-glycerophosphoric acid