title: Human endothelial and foetal femur-derived stem cell co-cultures modulate osteogenesis and angiogenesis.
abstract: BACKGROUND
  A dynamic vasculature is a prerequisite for bone formation where the interaction of bone cells and endothelial cells is essential for both the development and the healing process of bone. Enhanced understanding of the specific mediators involved in bone cell and endothelial cell interactions offers new avenues for skeletal regenerative applications. This study has investigated the osteogenic and angiogenic potential of co-cultures of human foetal diaphyseal or epiphyseal cells with human umbilical vein endothelial cells (HUVEC) in the presence and absence of vascular endothelial growth factor (VEGF) supplementation.
  
  
  METHODS
  Early osteogenic activities of the co-cultures (± VEGF) were assessed by alkaline phosphatase (ALP) activity. Osteogenic and angiogenic gene expression was measured using quantitative polymerase chain reaction. An ex vivo organotypic embryonic chick (E11) femur culture model was used to determine the osteogenic effects of VEGF as determined using micro-computed tomography (μCT) and Alcian blue/Sirius red histochemistry and immunocytochemistry for expression of CD31.
  
  
  RESULTS
  ALP activity and gene expression of ALP and Type-1 collagen was enhanced in foetal skeletal/HUVECs co-cultures. In foetal diaphyseal/HUVECs co-cultures, VEGF reduced the levels of ALP activity and displayed a negligible effect on von Willebrand factor (vWF) and VEGF gene expression. In contrast, VEGF supplementation was observed to significantly increase FLT-1 and KDR gene expression in co-cultures with modulation of expression enhanced, compared to VEGF skeletal monocultures. In the organotypic chick model, addition of VEGF significantly enhanced bone formation, which coincided with elevated levels of CD31-positive cells in the mid-diaphyseal region of the femurs.
  
  
  CONCLUSIONS
  These studies demonstrate a differential skeletal response of early foetal skeletal cells, when co-cultured with endothelial cells and the potential of co-culture models for bone repair. The differential effect of VEGF supplementation on markers of angiogenesis and osteogenesis in co-cultures and organ cultures, demonstrate the importance of the intricate temporal coordination of osteogenic and angiogenic processes during bone formation and implications therein for effective approaches to bone regenerative therapies.
authors.affiliation: Bone & Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Human Development and Health, Institute of Developmental Sciences, University of Southampton, Southampton, SO16 6YD, UK. s.inglis@soton.ac.uk.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Inglis
authors.affiliation: Human Development and Health, Centre for Human Development, Stem Cells and Regeneration, University of Southampton, Southampton, SO16 6YD, UK. dc1e10@soton.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Christensen
authors.affiliation: Human Development and Health, Centre for Human Development, Stem Cells and Regeneration, University of Southampton, Southampton, SO16 6YD, UK. D.I.Wilson@soton.ac.uk.
authors.forename: David I
authors.initials: DI
authors.lastname: Wilson
authors.affiliation: Bone & Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Human Development and Health, Institute of Developmental Sciences, University of Southampton, Southampton, SO16 6YD, UK. jk9@soton.ac.uk.
authors.forename: Janos M
authors.initials: JM
authors.lastname: Kanczler
authors.affiliation: Bone & Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Human Development and Health, Institute of Developmental Sciences, University of Southampton, Southampton, SO16 6YD, UK. roco@soton.ac.uk.
authors.forename: Richard O C
authors.initials: RO
authors.lastname: Oreffo
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D018920:Coculture Techniques; D024042:Collagen Type I / Q000378:metabolism; D066026:Endothelial Progenitor Cells / Q000502:physiology*; D005260:Female; D005269:Femur / Q000166:cytology; D015870:Gene Expression; D000066449:Human Embryonic Stem Cells / Q000502:physiology*; D006801:Humans; D018919:Neovascularization, Physiologic; D010012:Osteogenesis; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D024042:Collagen Type I; D042461:Vascular Endothelial Growth Factor A; D014841:von Willebrand Factor; C501162:FLT1 protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D000469:Alkaline Phosphatase