title: In vivo perfusion of human skin substitutes with microvessels formed by adult circulating endothelial progenitor cells.
abstract: BACKGROUND
  At present, tissue-engineered human skin substitutes (HSSs) mainly function as temporary bioactive dressings due to inadequate perfusion. Failure to form functional vascular networks within the initial posttransplantation period compromises cell survival of the graft and its long-term viability in the wound bed.
  
  
  OBJECTIVE
  Our goal was to demonstrate that adult circulating endothelial progenitor cells (EPCs) seeded onto HSS can form functional microvessels capable of graft neovascularization and perfusion.
  
  
  METHODS
  Adult peripheral blood mononuclear cells (PBMCs) underwent CD34 selection and endothelial cell (EC) culture conditions. After in vitro expansion, flow cytometry verified EC phenotype before their incorporation into HSS. After 2 weeks in vivo, immunohistochemical analysis, immunofluorescent microscopy, and microfil polymer perfusion were performed.
  
  
  RESULTS
  CD34+ PBMCs differentiated into EPC demonstrating characteristic EC morphology and expression of CD31, Tie-2, and E-selectin after TNFalpha-induction. Numerous human CD31 and Ulex europaeus agglutinin-1 (UEA-1) microvessels within the engineered grafts (HSS/EPCs) inosculated with recipient murine circulation. Limitation of murine CD31 immunoreactivity to HSS margins showed angiogenesis was attributable to human EPC at 2 weeks posttransplantation. Delivery of intravenous rhodamine-conjugated UEA-1 and microfil polymer to HSS/EPCs demonstrated enhanced perfusion by functional microvessels compared to HSS control without EPCs.
  
  
  CONCLUSIONS
  We successfully engineered functional microvessels in HSS by incorporating adult circulating EPCs. This autologous EC source can form vascular conduits enabling perfusion and survival of human bioengineered tissues.
authors.affiliation: Section of Dermatology, University of Chicago, Chicago, Illinois, USA. elaine.kung@uchospitals.edu
authors.forename: Elaine F
authors.initials: EF
authors.lastname: Kung
authors.forename: Feiya
authors.initials: F
authors.lastname: Wang
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Schechner
mesh_terms: D000818:Animals; D001808:Blood Vessels; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000637:transplantation; D004730:Endothelium, Vascular / Q000166:cytology*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000166:cytology*; D051379:Mice; D016513:Mice, SCID; D008856:Microscopy, Fluorescence; D018919:Neovascularization, Physiologic / Q000502:physiology*; D010477:Perfusion / Q000379:methods*; D033581:Stem Cell Transplantation / Q000379:methods; D013234:Stem Cells / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods*