title: Hypoxia impedes vasculogenesis of in vitro engineered bone.
abstract: To ensure the survival of engineered bone after implantation, we combined human endothelial colony forming cells (ECFCs) and multipotent stromal cells (MSCs) as a proof of concept in a co-culture model to create in vitro prevascularized bone constructs. We hypothesized that a hypoxic stimulus will contribute to prevascularization of engineered bone. Bone marrow-derived MSCs and ECFCs from human adult peripheral blood were allowed to form co-culture pellets containing ECFCs and MSCs (1:4) or MSCs only in controls. After culture under normoxia or hypoxia (5%), pellets were harvested and processed for immunohistochemistry of CD31, α-smooth muscle actin, and osteocalcin. Expression of vascular endothelial growth factor and SDF-1α was analyzed by PCR to elucidate their involvement in hypoxic stimulation of prevascularization. The normoxic condition in co-cultures of MSCs and ECFCs supported the formation and maintenance of prevascular structures, including organized CD31-positive cells embraced by differentiated mural cells. These structures failed to form in hypoxic conditions, thereby rejecting the hypothesis that hypoxia stimulates prevasculogenesis in three-dimensional engineered bone constructs. Further, the formation of prevascular structures was paralleled by increased SDF-1α expression. It is suggested that actual oxygen levels were below 5% in the hypoxic co-cultures, which prevented prevascular structure formation. In conclusion, our normoxic co-culture model containing cells from clinically relevant sources sustained simultaneous endothelial, smooth muscle, and osteogenic differentiation.
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands. d.gawlitta@umcutrecht.nl
authors.forename: Debby
authors.initials: D
authors.lastname: Gawlitta
authors.forename: Joost O
authors.initials: JO
authors.lastname: Fledderus
authors.forename: Mattie H P
authors.initials: MH
authors.lastname: van Rijen
authors.forename: Inge
authors.initials: I
authors.lastname: Dokter
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Alblas
authors.forename: Marianne C
authors.initials: MC
authors.lastname: Verhaar
authors.forename: Wouter J A
authors.initials: WJ
authors.lastname: Dhert
mesh_terms: D000328:Adult; D000368:Aged; D001596:Benzylamines; D015415:Biomarkers / Q000378:metabolism; D001842:Bone and Bones / Q000098:blood supply* / Q000187:drug effects / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects; D054377:Chemokine CXCL12 / Q000378:metabolism; D018920:Coculture Techniques; D003470:Culture Media / Q000494:pharmacology; D000080027:Cyclams; D005260:Female; D006571:Heterocyclic Compounds / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010012:Osteogenesis / Q000187:drug effects; D010100:Oxygen / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D001596:Benzylamines; D015415:Biomarkers; D054377:Chemokine CXCL12; D003470:Culture Media; D000080027:Cyclams; D006571:Heterocyclic Compounds; D010100:Oxygen; C088327:plerixafor