IADR Abstract Archives
Neutrophils enhance angiogenesis and osteogenesis in triple cell co-culture model
Objectives: Repair and regeneration of critical sized bone defects remain a major challenge in orthopaedic and maxillofacial surgery. This attempt has been hindered by inadequate vascularization of the deeper aspects of the implanted tissue construct. Considering foregoing information, aim of the present study was to establish a triple cell co-culture system using human neutrophils, osteoblasts and endothelial cells in vitro and examine the effect of neutrophils on osteogenesis and angiogenesis.
Methods: Primary osteoblasts (HOB) and endothelial cells (ECs) were co-cultured in the transwell system and the effect of neutrophils within triple-cell co-culture system was investigated using subsequent dose and time course experiments. The migration and homing of neutrophils were analysed using standard methodology such as FACS. The expression of osteogenic-specific and angiogenic-specific markers were analysed by gene array, qRT-PCR and ELISA. Histological examination was performed using specific osteogenic and angiogenic markers and the tubular network formation was assessed by immunocytochemistry, image analysis and matrigel assays.
Results: Neutrophil migration significantly induced the expression of angiogenic markers VEGF-A, EGF, FGF-2 and osteogenic markers ALP, OCN and OPN in co-culture model in dose and time related manner from 5 h to 15 days at RNA and protein level. Angiogenesis gene array analysis showed that neutrophils induced many genes related to growth factors and their receptors, chemokines and cytokines, matrix and adhesion molecules, as well as transcription factors. The formation of microvessel-like structures were detected in 7 days as well as 14 days in triple-cell co-culture model. Mineralisation content was gradually increased from 7 days to 14 days.
Conclusions: This pioneering triple-cell co-culture model demonstrated that neutrophils could enhance angiogenesis and osteogenesis, an invaluable novel insight. This novel strategy for bone regeneration and enhancing microvasculature, may be applied clinically in the field of oral and maxillofacial surgery, bringing enormous benefit to patients.
Methods: Primary osteoblasts (HOB) and endothelial cells (ECs) were co-cultured in the transwell system and the effect of neutrophils within triple-cell co-culture system was investigated using subsequent dose and time course experiments. The migration and homing of neutrophils were analysed using standard methodology such as FACS. The expression of osteogenic-specific and angiogenic-specific markers were analysed by gene array, qRT-PCR and ELISA. Histological examination was performed using specific osteogenic and angiogenic markers and the tubular network formation was assessed by immunocytochemistry, image analysis and matrigel assays.
Results: Neutrophil migration significantly induced the expression of angiogenic markers VEGF-A, EGF, FGF-2 and osteogenic markers ALP, OCN and OPN in co-culture model in dose and time related manner from 5 h to 15 days at RNA and protein level. Angiogenesis gene array analysis showed that neutrophils induced many genes related to growth factors and their receptors, chemokines and cytokines, matrix and adhesion molecules, as well as transcription factors. The formation of microvessel-like structures were detected in 7 days as well as 14 days in triple-cell co-culture model. Mineralisation content was gradually increased from 7 days to 14 days.
Conclusions: This pioneering triple-cell co-culture model demonstrated that neutrophils could enhance angiogenesis and osteogenesis, an invaluable novel insight. This novel strategy for bone regeneration and enhancing microvasculature, may be applied clinically in the field of oral and maxillofacial surgery, bringing enormous benefit to patients.