Promotion of angiogenesis in vitro by Astragalus polysaccharide via activation of TLR4 signaling pathway

During the implantation of functional tissue‐engineered constructs for treating bone defects, a functional vascular network is critical for the survival of the construct. One strategy to achieve rapid angiogenesis for this application is the co‐culture of outgrowth endothelial cells (OECs) and primary human osteoblasts (POBs) within a scaffold prior to implantation. In the present study, we aim to investigate whether Astragalus polysaccharide (APS) promotes angiogenesis or vascularization via the TLR4 signaling pathway in a co‐culture of OECs and POBs. The co‐cultures were treated with various concentrations of APS for 24 h and, subsequently, another 7 days, followed by CD31 staining and analysis of micro‐vessel‐formation areas using software. Additionally, APS (0.4 mg/ml for 24 h) was added to monocultures of OECs or POBs for evaluating proliferation, apoptosis, angiogenesis, osteogenesis, TLR4 signaling pathway, and inflammatory cytokine release. We found that APS promoted angiogenesis in the co‐culture at the optimal concentration of 0.4 mg/ml. TLR4 activation by APS up‐regulated the expression level of TLR4/MyD88 and enhanced angiogenesis and osteogenesis in monocultures of OECs and POBs. The levels of E‐selectin adhesion molecules, three cytokines (IL‐6, TNF‐α, and IFN‐γ), and VEGF and PDGF‐BB, which can induce angiogenesis, increased significantly (p