Investigation Of Macrophage Polarization Mediated By Chitooligosaccharide (COS) And Associated Osteogenic And Angiogenic Activities

The host response to implanted biomaterials can influence the functionality of the materials and modulate the tissue repair and remolding.Macrophages,key cells in the host response to biomaterials,can be polarized into different phenotypes,which are important in regenerative medicine.More research have shown that chitosan has a good biological compatibility,and is an agent good for immune,inflammation and bone repair regulation.Chitosan based biomaterials have been widely used in clinical research of bone regeneration.However,it is not clear how chitosan based biomaterials exert effects on the body after implantation,and the specific effects of their degradation products.Therefore,it is of great significance to study the mechanism of degradation products of chitosan based biomaterials in the body for the extensive application.The objective of this study was to evaluate the effect of chitooligosaccharide(COS)on the modulation of macrophage(RAW 264.7)polarization and the associated osteogenic and angiogenic activities.The results demonstrate that COS can shift the macrophage response to an M2 reparative response,which can then upregulate the expression of anti-inflammatory cytokines.COS can also create an immune-modulated microenvironment,with osteogenesis-and angiogenesis-related proteins and a biological process that further influences the osteogenic/angiogenic differentiation and promotion of bone mesenchymal stem cells(BMSCs)and vascular activation of human umbilical vein endothelial cells(HUVECs).In this work,COS at a low concentration of 4 ?g/mL(COS(4))and suitable polymerization degrees of 5(Chitopentaose Hydrochloride,COS5),the associated effect on an M2 reparative response and upregulation of anti-inflammatory cytokines expression was better than COS at other concentrations or polymerization degrees.The supernatant from a culture of RAW 264.7 stimulated by COS(4)and COS5(conditioned medium S-COS(4)and S-COS5),contain more osteogenesis-and angiogenesis-related proteins like DKK-1,OPN,Osteoactivin,and VEGF R1,EGF,IGFBP-5 for positive regulation of osteogenesis/angiogenesis.Specifically,the alkaline phosphatase activity(ALP)and typical osteogenesis-related proteins of BMSCs were significantly influenced by the conditioned media of COS-stimulated macrophages(S-COS(4)and S-COS5).Furthermore,the conditioned media promoted HUVEC proliferation and migration for vascularization.Further,to better simulate the in vivo environment,macrophages and stem cell/ endothelial cells were co-cultured in a Transwell chamber,to verify macrophages polarization for associated osteogenic and angiogenic activities.When cocultured with RAW264.7 in COS(4)and COS5 media respectively,the alkaline phosphatase activity(ALP)and typical osteogenesis-related proteins or genes of BMSCs were upregulated,and the proliferation,migration activity and angiogenesis related processes of endothelial cells were improved.Our results suggest that COS at a low concentration and suitable polymerization degrees has a beneficial effect on immunity modulation(an M2 reparative response),and can modulate osteogenesis/angiogenesis processes for tissue regeneration without using any inductive agent.