1.温州医科大学 病理学与病理生理学教研室，浙江 温州 325035；2.University of Western Australia，Perth WA6009；3.温州医科大学附属第一医院 骨科，浙江 温州 325015；4.温州医科大学 第一临床医学院，浙江 温州 325035；5.温州医科大学附属第二医院育英儿童医院 病理科，浙江 温州 325027
Vascularization mechanism of periosteal decellularized bioscaffold implanted in bone defect mice
ZHANG Xueming1, CHEN Junhao2, CHEN Lei3, LI Xiaohang4, WANG Kaicheng1, LIN Qiongqiong5, JIN Keke1
1.Department of Pathophysiology, Wenzhou Medical University, Wenzhou 325035, China; 2.University of Western Australia, Perth WA6009, Australia; 3.Department of Orthopedics, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China; 4.The First Clinical Medical College, Wenzhou Medical University, Wenzhou 325035, China; 5.Department of Pathology, the Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China
ZHANG Xueming,CHEN Junhao,CHEN Lei, et al. Vascularization mechanism of periosteal decellularized bioscaffold implanted in bone defect mice[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2019, 49(5): 350-355.
Abstract：Objective: To observe the reparative effect of periosteal decellularized bioscaffold on bone defect in mice and the process of angiogenesis in the scaffold and to elucidate the possible mechanisms underlying the angiogenesis. Methods: The periosteal decellularized biological scaffolds were obtained using sequential treatment including physical freeze-thaw, chemical and biological enzyme reagents processing. To investigate in vitro effects of periosteal decellularized scaffold extract on human umbilical vein endothelial cells (HUVECs), wound healing assay was used to evaluate the presence of pro-vascularized biological factors in scaffolds; Meanwhile, Enzyme-linked immunosorbent assay (ELISA) was used to detect vascular endothelial growth factor (VEGF) in scaffolds. The bone reparative ability of the periosteal decellularized bioscaffold due to its pro-vascularization effect was further evaluated in a mouse femur bone defect model. A 0.5 mm diameter single cortical bone defect was prepared at the distal femur of the mouse, in which the periosteal decellularized scaffold was implanted, followed by layer-by-lay suture. The bone defects without material implanted were set as the control group. On the day 7, 14, 21 and 28 after surgery, the materials were harvested, fixed, decalcified, embedded, and sectioned. The bone repair was evaluated by HE staining. Immunofluorescence staining of von Willebrand Factor (vWF) was performed to observe the vascularization within the defect area. Results: Cell experiments showed that the decellularized periosteum scaffold extract had no obvious inhibitory effect on the proliferation of HUVEC. The results of cell wound healing test showed that the migration area of the scaffold extract group was larger than that of the control group, suggesting that the decellularized periosteum scaffold extract can effectively promote the migration of HUVEC. Moreover, VEGF was present in the scaffold extract at a concentration of 210 pg/mL. In animal experiments, HE staining demonstrated that decellularized periosteal scaffolds can promote the growth of blood vessels and new bone formation in the bone defect area. Immunofluorescence staining further proved that the repair activity of the decellularized periosteum scaffold was associated with the process of vascularization, and the density of blood vessels in the decellularized periosteal scaffold increased first and then decreased. Conclusion: Decellularized periosteal scaffolds can vascularize and promote healing of bone defects. VEGF may be a key factor in its angiogenesis process.
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