title: Angiogenesis in newly regenerated bone by secretomes of human mesenchymal stem cells.
abstract: BACKGROUND
  For an effective bone graft for reconstruction of the maxillofacial region, an adequate vascular network will be required to supply blood, osteoprogenitor cells, and growth factors. We previously reported that the secretomes of bone marrow-derived mesenchymal stem cells (MSC-CM) contain numerous growth factors such as insulin-like growth factor (IGF)-1, transforming growth factor (TGF)-β1, and vascular endothelial growth factor (VEGF), which can affect the cellular characteristics and behavior of regenerating bone cells. We hypothesized that angiogenesis is an important step for bone regeneration, and VEGF is one of the crucial factors in MSC-CM that would enhance its osteogenic potential. In the present study, we focused on VEGF in MSC-CM and evaluated the angiogenic and osteogenic potentials of MSC-CM for bone regeneration.
  
  
  METHODS
  Cytokines in MSC-CM were measured by enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) were cultured with MSC-CM or MSC-CM with anti-VEGF antibody (MSC-CM + anti-VEGF) for neutralization, and tube formation was evaluated. For the evaluation of bone and blood vessel formation with micro-computed tomography (micro-CT) and for the histological and immunohistochemical analyses, a rat calvarial bone defect model was used.
  
  
  RESULTS
  The concentrations of IGF-1, VEGF, and TGF-β1 in MSC-CM were 1515.6 ± 211.8 pg/mL, 465.8 ± 108.8 pg/mL, and 339.8 ± 14.4 pg/mL, respectively. Tube formation of HUVECs, bone formation, and blood vessel formation were increased in the MSC-CM group but decreased in the MSC-CM + anti-VEGF group. Histological findings suggested that new bone formation in the entire defect was observed in the MSC-CM group although it was decreased in the MSC-CM + anti-VEGF group. Immunohistochemistry indicated that angiogenesis and migration of endogenous stem cells were much more abundant in the MSC-CM group than in the MSC-CM + anti-VEGF group.
  
  
  CONCLUSIONS
  VEGF is considered a crucial factor in MSC-CM, and MSC-CM is proposed to be an adequate therapeutic agent for bone regeneration with angiogenesis.
authors.affiliation: Division of Reconstructive Surgery for Oral and Maxillofacial Region, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.|Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Aichi, Japan.
authors.forename: Wataru
authors.identifier: 0000-0002-0444-3314
authors.initials: W
authors.lastname: Katagiri
authors.affiliation: Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Aichi, Japan.
authors.forename: Takamasa
authors.initials: T
authors.lastname: Kawai
authors.affiliation: Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Aichi, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Osugi
authors.affiliation: Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Aichi, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Sugimura-Wakayama
authors.affiliation: Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Aichi, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Sakaguchi
authors.affiliation: Division of Reconstructive Surgery for Oral and Maxillofacial Region, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Kojima
authors.affiliation: Division of Reconstructive Surgery for Oral and Maxillofacial Region, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Tadaharu
authors.initials: T
authors.lastname: Kobayashi
keywords: Angiogenesis; Bone; Conditioned medium; Cytokine; Mesenchymal stem cells; Secretome