Resveratrol and Angiogenin-2 Combined With PEGDA/TCS Hydrogel for the Targeted Therapy of Hypoxic Bone Defects via Activation of the Autophagy Pathway.

Dehui Fan,Zhixian Yuan,Meiyi Su,Wenqiang Li,Shuquan Yang,Ying Lin,Zhenning Zhang,Xintao Zhang,Hengping Liu

doi:10.3389/fphar.2021.618724

Abstract

The guarantee of cell survival under hypoxic conditions and rapid vascularization is a key in tissue engineering strategies for treating bone defects. Our study aimed to establish the protective role of bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) in hypoxic conditions and realize rapid vascularization in bone defects. Resveratrol (Res), a non-flavonoid polyphenolic compound, and angiopoietin-2 (ANG2), a vascular activating factor, were applied to enhance BMSC and HUVEC survival, osteogenesis, and angiogenesis. The morphology, autophagy, viability, apoptosis, cycle, and osteogenic differentiation of BMSCs treated with Res were analyzed. The results indicated that Res could improve BMSC survival and differentiation via the autophagy pathway under hypoxic conditions. In addition, Res maintained HUVEC growth and proliferation in a hypoxic and ANG2 double-adverse environment via the autophagy pathway. To simulate a relatively hypoxic environment, small-aperture PEGDA/TCS hydrogels containing Res and ANG2 were prepared. BMSCs were cultured in the PEGDA/TCS scaffold and transplanted into a large tibial defect. CD31 immunofluorescence showed that the density and size of new blood vessels in the bone defect were significantly enhanced by ANG2 and Res at 8 weeks after surgery. H&E, Masson, and immunohistochemical staining results indicated that ANG2 combined with Res could promote new bone formation in defects. All these results suggested that Res combined with ANG2 may be a novel strategy for the targeted therapy of hypoxic bone defects with tissue engineering scaffolds.

Highlights

Bone defect reconstruction is a continuous and complex biological process involving many functional cells, the extracellular matrix and various factors (Drosse et al, 2008; Horner et al, 2010)
In the in vitro experiment, ROS detection, cell viability, flow cytometry and AO-EB staining indicated that Res protected bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) against cell damage and apoptosis by activating autophagy in the hypoxic environment
In the in vivo experiment, Res and ANG2 were cultured in PEGDA/Thiolated chitosan (TCS) scaffolds and transplanted into the large bone defect area

Summary

INTRODUCTION

Bone defect reconstruction is a continuous and complex biological process involving many functional cells, the extracellular matrix and various factors (Drosse et al, 2008; Horner et al, 2010). Hydrogels with small apertures and excellent biocompatibility were prepared to simulate relatively hypoxic PEGDA/TCS tissue engineering scaffolds After they were loaded with Res and ANG2 and co-cultured with BMSCs in vitro, they were implanted into a large-segment tibial bone defect in rats to investigate the ability of Res and ANG2 in repairing largesegment bone defects by using histology, immunohistochemistry, and immunofluorescence. BMSCs were treated with 3-MA, Res, and Res/3-MA at final concentrations of 5 mM, 50 μg/ml, and 50 μg/ml/5 mM for 10 h in a six-well plate After they were washed and centrifuged, the harvested cells were stained with an Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit for 15 min at room temperature and analyzed by a flow cytometer. Differences were considered significant at *p < 0.05 and **p < 0.01

RESULTS AND DISCUSSION

CONCLUSION

ETHICS STATEMENT