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  • Title: [Establishment and biological effect evaluation of prevascularized porous β-tricalcium phosphate tissue engineered bone].
    Author: Huang M, Fan J, Ma Z, Li J, Lu Y.
    Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2022 May 15; 36(5):625-632. PubMed ID: 35570639.
    Abstract:
    OBJECTIVE: To evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. METHODS: EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group ( n=6) and porous β-TCP scaffold as the control group ( n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. RESULTS: The isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel ( P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group ( P<0.05), except for area fraction at 4 weeks after implantation ( P>0.05). CONCLUSION: The prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair. 目的: 基于组织工程技术，采用内皮祖细胞（endothelial progenitor cells，EPCs）和BMSCs共培养方式构建预血管化多孔β-磷酸三钙（β-tricalcium phosphate，β-TCP）组织工程骨（以下简称预血管化组织工程骨），并评价其对骨修复过程中血管化的影响。. 方法: 取新西兰大白兔髂骨骨髓，采用密度梯度离心法和差速贴壁法分离EPCs和BMSCs并传代，经细胞免疫表型检测、BMSCs诱导分化和EPCs吞噬功能鉴定后，取第3代细胞进行后续实验。首先，采用Matrigel基质胶成管实验检测体外EPCs/BMSCs共培养（EPCs/BMSCs组）成管情况，以单纯EPCs（EPCs组）作为对照；然后，取多孔β-TCP生物陶瓷支架与EPCs/BMSCs共培养7 d构建预血管化组织工程骨（EPCs/BMSCs组），以与单纯EPCs共培养支架（EPCs组）作为对照，扫描电镜和激光共聚焦显微镜观察细胞在支架上黏附、增殖及成管情况；最后，取12只新西兰大白兔制备股骨髁缺损模型，分别植入预血管化组织工程骨（实验组， n=6）以及多孔β-TCP生物陶瓷支架（对照组， n=6），通过Microfill血管灌注、Micro-CT扫描、荧光背景下血管成像方法观察术后4、8周支架内部血管化进程并定量评估血管数量、血管直径和面积分数。. 结果: 经鉴定分离培养的细胞为BMSCs和EPCs。两种细胞共培养后逐渐形成管型样结构；培养6 h时EPCs/BMSCs组在Matrigel基质胶形成管型样结构数量、分支数量和成管总长度均优于EPCs组（ P<0.05）。细胞种植至支架上培养7 d后，EPCs组细胞形成膜片结构贴附在支架上，EPCs/BMSCs组细胞贴附更紧密、细胞膜片更厚、细胞数量和形成的管型样结构更多。植入动物体内4周，两组支架内部均有新生血管长入，8周新生血管均出现了改建。除术后4周两组面积分数差异无统计学意义（ P>0.05）外，其余各时间点实验组血管数量、血管直径以及面积分数均优于对照组，差异有统计学意义（ P<0.05）。. 结论: 基于BMSCs和EPCs直接接触共培养构建预血管化组织工程骨，用于骨缺损修复可显著促进早期血管化进程。.
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