title: Endothelial microvascular networks affect gene-expression profiles and osteogenic potential of tissue-engineered constructs.
abstract: BACKGROUND
  A major determinant of the potential size of cell/scaffold constructs in tissue engineering is vascularization. The aims of this study were twofold: first to determine the in vitro angiogenic and osteogenic gene-expression profiles of endothelial cells (ECs) and mesenchymal stem cells (MSCs) cocultured in a dynamic 3D environment; and second, to assess differentiation and the potential for osteogenesis after in vivo implantation.
  
  
  METHODS
  MSCs and ECs were grown in dynamic culture in poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) copolymer scaffolds for 1 week, to generate three-dimensional endothelial microvascular networks. The constructs were then implanted in vivo, in a murine model for ectopic bone formation. Expression of selected genes for angiogenesis and osteogenesis was studied after a 1-week culture in vitro. Human cell proliferation was assessed as expression of ki67, whereas α-smooth muscle actin was used to determine the perivascular differentiation of MSCs. Osteogenesis was evaluated in vivo through detection of selected markers, by using real-time RT-PCR, alkaline phosphatase (ALP), Alizarin Red, hematoxylin/eosin (HE), and Masson trichrome staining.
  
  
  RESULTS
  The results show that endothelial microvascular networks could be generated in a poly(LLA-co-DXO) scaffold in vitro and sustained after in vivo implantation. The addition of ECs to MSCs influenced both angiogenic and osteogenic gene-expression profiles. Furthermore, human ki67 was upregulated before and after implantation. MSCs could support functional blood vessels as perivascular cells independent of implanted ECs. In addition, the expression of ALP was upregulated in the presence of endothelial microvascular networks.
  
  
  CONCLUSIONS
  This study demonstrates that copolymer poly(LLA-co-DXO) scaffolds can be prevascularized with ECs and MSCs. Although a local osteoinductive environment is required to achieve ectopic bone formation, seeding of MSCs with or without ECs increases the osteogenic potential of tissue-engineered constructs.
authors.forename: Torbjorn O
authors.initials: TO
authors.lastname: Pedersen
authors.forename: Anna L
authors.initials: AL
authors.lastname: Blois
authors.forename: Zhe
authors.initials: Z
authors.lastname: Xing
authors.forename: Ying
authors.initials: Y
authors.lastname: Xue
authors.forename: Yang
authors.initials: Y
authors.lastname: Sun
authors.forename: Anna
authors.initials: A
authors.lastname: Finne-Wistrand
authors.forename: Lars A
authors.initials: LA
authors.lastname: Akslen
authors.forename: James B
authors.initials: JB
authors.lastname: Lorens
authors.forename: Knut N
authors.initials: KN
authors.lastname: Leknes
authors.forename: Inge
authors.initials: I
authors.lastname: Fristad
authors.forename: Kamal
authors.initials: K
authors.lastname: Mustafa
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation; D002454:Cell Differentiation; D018920:Coculture Techniques; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D019394:Ki-67 Antigen / Q000378:metabolism; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D055806:Microvessels / Q000166:cytology* / Q000378:metabolism; D010012:Osteogenesis; D011091:Polyesters / Q000737:chemistry; D023822:Tissue Engineering; D054457:Tissue Scaffolds; D059467:Transcriptome; D014183:Transplantation, Heterologous
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D019394:Ki-67 Antigen; D011091:Polyesters; C556909:poly(L-lactide)-co-(1,5-dioxepan-2-one)