Angiogenesis of 2-N, 6-O-sulfated Chitosan and its Effect on the Vascularization During Bone Regeneration

Objectives: Angiogenesis, or neovascularization, is a critical factor for regenerative bone tissue, because the existence of a functional vascular network within the defect site can provide sufficient oxygen and nutrients to facilitate growth, differentiation, and tissue functionality. In the present study, 2-N, 6-O-sulfated chitosan (26SCS) as a kind of heparin-like polysaccharide was hypothesized to regulate cell behavior of HUVECs and achieve more enhanced vessel formation thereafter more effective bone regeneration in vivo.
Methods: We first synthesized and characterized 26SCS, and then assessed its effects on cell proliferation, adhesion of human umbilical vein endothelial cells (HUVECs). Afterwards, we determined the effect of 26SCS on gene expression of angiogenesis-related cytokine by HUVECs. Furthermore, we investigated the effect of 26SCS on the vascularization during the ectopic and orthotopic bone formation.
Results: The molecular structure of 26SCS was confirmed by FTIR and ¹³C NMR spectra. Compared to heparin, concentration of 26SCS had enhancement effect on cell viability of HUVECs at 0-0.08 μg/mL dose dependently. HUVECs cultured with 0.08 μg/mL 26SCS showed increasing numbers and significantly more outstretched filopodia-like extensions. According to the assay for endothelial markers’ gene expressions, 26SCS could up-regulate the expression of ANG of HUVECs significantly (p < 0.05) at 0.08 μg/mL while up-regulate FGF-2 significantly at 0.04 μg/mL. We evaluated in vivo ectopic bone formation induced by complex of rhBMP-2 with a gradient levels of 26SCS placed in the thigh muscle pouches of mice 4 weeks post-implantation, respectively. New bone formation was found rising with increase of 26SCS dose. 26SCS/50 group showed a larger area of positive expression of CD31. Similar results were found when rhBMP-2/26SCS complex implants were implanted to the segmental defect of the rabbit radius.
Conclusions: Our research verified clear advantages of 26SCS for enhancing angiogenesis in bone formation. This polymer could be used to help rebuild the vessel network during bone repair. In addition, the polymer might also be used to help the delivery of a series of growth factors, which have affinity with heparin.