骨膜去细胞生物支架在骨缺损小鼠体内的血管化机制
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摘要
目的:观察骨膜去细胞生物支架对小鼠股骨骨缺损的修复作用及支架内血管形成过程,探讨其血管形成的可能机制。方法:采用物理冻融、化学和生物酶试剂等序贯处理获得骨膜去细胞生物支架。体外实验方面,通过细胞划痕试验观察骨膜去细胞支架浸提液对人脐静脉内皮细胞(HUVEC)迁移的影响以评价骨膜去细胞支架中是否存在促血管化的生物因子,同时通过酶联免疫吸附试验(ELISA)检测支架中的血管内皮生长因子(VEGF)。在体动物实验方面,通过建立小鼠股骨骨缺损模型评价骨膜去细胞生物支架通过促血管化诱导骨修复的可能性。在小鼠股骨远端制备0.5mm直径的单皮质骨缺损后,于骨缺损处植入骨膜去细胞支架后逐层缝合切口,对照组小鼠骨缺损处不放置材料。分别在术后第7天、第14天、第21天和第28天,取材、固定、脱钙、包埋和切片,通过HE染色评价骨缺损区骨修复情况,通过免疫荧光染色观察血管性血友病因子(vWF)以评价缺损区血管化情况。结果:体外细胞实验表明,去细胞骨膜支架浸提液对HUVEC的增殖没有明显的抑制作用;细胞划痕实验结果显示,与对照组相比,支架浸提液组细胞的迁移面积更大,去细胞骨膜支架浸提液能够有效促进HUVEC的迁移并且在支架浸提液中检测到VEGF,其浓度为210 pg/m L。动物实验方面,HE染色证明去细胞骨膜支架可以促进骨缺损区域血管的生长和新骨形成,免疫荧光染色进一步证明去细胞骨膜支架对骨缺损的修复活动伴随血管化的发生过程,且去细胞骨膜支架中血管的密度随时间延长呈现先增多后减小的趋势。结论:去细胞骨膜支架可以血管化,并且促进骨缺损的愈合。VEGF可能是其血管形成过程中的关键因素。
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 provascularization 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.
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 provascularization 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.
引文
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[13]WAGNER E R,PARRY J,DADSETAN M,et al.VEGF-mediated angiogenesis and vascularization of a fumaratecrosslinked polycaprolactone(PCLF)scaffold[J].Connect Tissue Res,2018,59(6):542-549.
[14]HU K,OLSEN B R.Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair[J].J Clin Invest,2016,126(2):509-526.
[2]ZHANG X,AWAD H A,O'KEEFE R J,et al.A perspective:engineering periosteum for structural bone graft healing[J].Clin Orthop Relat R,2008,466(8):1777-1787.
[3]CHEN K,LIN X,ZHANG Q,et al.Decellularized periosteum as a potential biologic scaffold for bone tissue engineering[J].Acta Biomater,2015,19(6):46-55.
[4]黄俊杰,王志斌,陈纳,等.大网膜包埋移植肝脏去细胞生物支架血管化过程观察[J].温州医科大学学报,2017,47(10):703-707.
[5]LI T,PENG M,YANG Z,et al.3D-printed IFN-γ-loading calcium silicate-β-tricalcium phosphate scaffold sequentially activates M1 and M2 polarization of macrophages to promote vascularization of tissue engineering bone[J].Acta Biomater,2018,71:96-107.
[6]SALCEDO R,WASSERMAN K,YOUNG H A,et al.Vascular endothelial growth factor and basic fibroblast growth factor induce expression of CXCR4 on human endothelial cells:In vivo neovascularization induced by stromal-derived factor-1alpha[J].Am J Pathol,1999,154(4):1125-1135.
[7]ZHANG G W,GU T X,GUAN X Y,et al.bFGF binding cardiac extracellular matrix promotes the repair potential of bone marrow mesenchymal stem cells in a rabbit model for acute myocardial infarction[J].Biomed Mater,2015,10(6):065018.
[8]周敏,刘长建,卫志庆,等.bFGF预载去细胞支架构建小口径人工血管的研究[J].中国修复重建外科杂志,2008,22(3):370-375.
[9]XIANG Y,HWA J.Regulation of VWF expression,and secretion in health and disease[J].Curr Opin Hematol,2016,23(3):288-293.
[10]ALBERTI K A,XU Q.Biocompatibility and degradation of tendon-derived scaffolds[J].Regen Biomater,2016,3(1):1-11.
[11]MEI J,YU Y,LI M,et al.The angiogenesis in decellularized scaffold-mediated the renal regeneration[J].Oncotarget,2016,7(19):27085-27093.
[12]COULTAS L,CHAWENGSAKSOPHAK K,ROSSANT J.Endothelial cells and VEGF in vascular development[J].Nature,2005,438(7070):937-945.
[13]WAGNER E R,PARRY J,DADSETAN M,et al.VEGF-mediated angiogenesis and vascularization of a fumaratecrosslinked polycaprolactone(PCLF)scaffold[J].Connect Tissue Res,2018,59(6):542-549.
[14]HU K,OLSEN B R.Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair[J].J Clin Invest,2016,126(2):509-526.