IADR Abstract Archives
GM-CSF Activated Neutrophils Induce Neovascularization in Triple Cell co-Culture Model
Objectives: Neovascularization is an important part of bone tissue engineering. Inadequate neovascularization hinders proper transport of nutrients and oxygen supply to the bone graft, resulting in failure of the bone regeneration. Previously we demonstrated that neutrophils enhance angiogenesis and osteogenesis in a triple cell co-culture model. However, the major limitation factor for practical application of neutrophils is their short lifespan. Granulocyte macrophage colony-stimulating factor (GM-CSF) has shown to enhance the survival of neutrophils. The objective of the present study was to investigate the efficacy of GM-CSF activated neutrophils to enhance angiogenesis in bone regeneration.
Methods: Human neutrophils were isolated from peripheral blood according to a previously optimized protocol. Primary osteoblasts (HOB) and endothelial cells (HUVECs) were co-cultured in the transwell system and the effect of GM-CSF activated neutrophils was examined by dose- and time-dependent assays using different doses of 0.5-100ng/ml GM-CSF for 4-72 h. In-vitro angiogenic potential of GM-CSF activated neutrophils was evaluated on proliferation, expression of angiogenic markers and microvessel formation by cell-counting, qRT-PCR, ELISA and confocal immunofluorescence assays respectively.
Results: GM-CSF activated neutrophils significantly up regulated the gene expression levels of pro-inflammatory marker IL-8, basic angiogenic markers VEGF-2, EGF, FGF-2 and MMP-9 compared to the non-activated neutrophils in dose- and time- dependent manner (P<0.05). Moreover, the protein expression levels of VEGF-2 and MMP-9 were significantly induced by GM-CSF activated neutrophils compared to controls (P<0.05). Notably, activated neutrophils accelerated proliferative capacity of HUVECs in a time course study. Interestingly, formation of microvessel-like structures was comparatively increased by activated neutrophils as indicated by CD31 expression at 14 days.
Conclusions: This study provided a new insight on the positive activity of GM-CSF activated neutrophils on neovascularization in bone regeneration. This novel strategy to enhance microvasculature may bring significant advantages in the field of bone tissue engineering, particularly for treating bone defects in oral-maxillofacial region.
Methods: Human neutrophils were isolated from peripheral blood according to a previously optimized protocol. Primary osteoblasts (HOB) and endothelial cells (HUVECs) were co-cultured in the transwell system and the effect of GM-CSF activated neutrophils was examined by dose- and time-dependent assays using different doses of 0.5-100ng/ml GM-CSF for 4-72 h. In-vitro angiogenic potential of GM-CSF activated neutrophils was evaluated on proliferation, expression of angiogenic markers and microvessel formation by cell-counting, qRT-PCR, ELISA and confocal immunofluorescence assays respectively.
Results: GM-CSF activated neutrophils significantly up regulated the gene expression levels of pro-inflammatory marker IL-8, basic angiogenic markers VEGF-2, EGF, FGF-2 and MMP-9 compared to the non-activated neutrophils in dose- and time- dependent manner (P<0.05). Moreover, the protein expression levels of VEGF-2 and MMP-9 were significantly induced by GM-CSF activated neutrophils compared to controls (P<0.05). Notably, activated neutrophils accelerated proliferative capacity of HUVECs in a time course study. Interestingly, formation of microvessel-like structures was comparatively increased by activated neutrophils as indicated by CD31 expression at 14 days.
Conclusions: This study provided a new insight on the positive activity of GM-CSF activated neutrophils on neovascularization in bone regeneration. This novel strategy to enhance microvasculature may bring significant advantages in the field of bone tissue engineering, particularly for treating bone defects in oral-maxillofacial region.