title: Cell sheet engineering using the stromal vascular fraction of adipose tissue as a vascularization strategy.
abstract: Current vascularization strategies for Tissue Engineering constructs, in particular cell sheet-based, are limited by time-consuming and expensive endothelial cell isolation and/or by the complexity of using extrinsic growth factors. Herein, we propose an alternative strategy using angiogenic cell sheets (CS) obtained from the stromal vascular fraction (SVF) of adipose tissue that can be incorporated into more complex constructs. Cells from the SVF were cultured in normoxic and hypoxic conditions for up to 8days in the absence of extrinsic growth factors. Immunocytochemistry against CD31 and CD146 revealed spontaneous organization in capillary-like structures, more complex after hypoxic conditioning. Inhibition of HIF-1α pathway hindered capillary-like structure formation in SVF cells cultured in hypoxia, suggesting a role of HIF-1α. Moreover, hypoxic SVF cells showed a trend for increased secretion of angiogenic factors, which was reflected in increased network formation by endothelial cells cultured on matrigel using that conditioned medium. In vivo implantation of SVF CS in a mouse hind limb ischemia model revealed that hypoxia-conditioned CS led to improved restoration of blood flow. Both in vitro and in vivo data suggest that SVF CS can be used as simple and cost-efficient tools to promote functional vascularization of TE constructs.
  
  
  
  Neovascularization after implantation is a major obstacle for producing clinically viable cell sheet-based tissue engineered constructs. Strategies using endothelial cells and extrinsic angiogenic growth factors are expensive and time consuming and may raise concerns of tumorigenicity. In this manuscript, we describe a simplified approach using angiogenic cell sheets fabricated from the stromal vascular fraction of adipose tissue. The strong angiogenic behavior of these cell sheets, achieved without the use of external growth factors, was further stimulated by low oxygen culture. When implanted in an in vivo model of hind limb ischemia, the angiogenic cell sheets contributed to blood flux recovery. These cell sheets can therefore be used as a straightforward tool to increase the neovascularization of cell sheet-based thick constructs.
authors.affiliation: 3B's Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4805-017 Barco, Guimarães, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal; Institute of Biophysics and Biomedical Engineering, Faculty of Sciences of the University of Lisbon, Lisbon, Portugal.
authors.forename: Marina
authors.initials: M
authors.lastname: Costa
authors.affiliation: 3B's Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4805-017 Barco, Guimarães, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.
authors.forename: Mariana T
authors.initials: MT
authors.lastname: Cerqueira
authors.affiliation: 3B's Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4805-017 Barco, Guimarães, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.
authors.forename: Tírcia C
authors.initials: TC
authors.lastname: Santos
authors.affiliation: Institute of Life and Health Sciences, School of Medicine, University of Minho, Braga, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.
authors.forename: Belém
authors.initials: B
authors.lastname: Sampaio-Marques
authors.affiliation: Institute of Life and Health Sciences, School of Medicine, University of Minho, Braga, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.
authors.forename: Paula
authors.initials: P
authors.lastname: Ludovico
authors.affiliation: 3B's Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4805-017 Barco, Guimarães, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.
authors.forename: Alexandra P
authors.initials: AP
authors.lastname: Marques
authors.affiliation: 3B's Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4805-017 Barco, Guimarães, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.
authors.forename: Rogério P
authors.initials: RP
authors.lastname: Pirraco
authors.affiliation: 3B's Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4805-017 Barco, Guimarães, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal. Electronic address: rgreis@dep.uminho.pt.
authors.forename: Rui L
authors.initials: RL
authors.lastname: Reis
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation; D000328:Adult; D000818:Animals; D015687:Cell Hypoxia; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation; D005260:Female; D064593:Heterografts; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D008297:Male; D051379:Mice; D018919:Neovascularization, Physiologic; D023822:Tissue Engineering
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit
keywords: Adipose tissue; Cell sheet engineering; Hypoxia; SVF; Vascularization