人骨髓间充质干细胞/细胞外基质复合体用于骨缺损再生修复的研究

Human Bone Marrow Mesenchymal Stem Cells/Extracellular Matrix Complex for Bone Defect Regeneration and Repair

目的:评估人骨髓间充质干细胞/细胞外基质复合体(C-BMSC)的骨缺损再生修复能力。方法:体外构建C-BMSC,通过大体观察、HE染色、扫描电镜进行组织形态学检测;TUNEL染色法检测细胞凋亡水平;免疫荧光染色和PCR法检测成骨相关因子的表达水平;大鼠颅骨缺损模型评估C-BMSC体内骨缺损再生修复能力。结果:组织形态学显示5 d组C-BMSC的细胞外基质(ECM)最丰富,ECM将细胞完全包裹,ECM纤维粗壮,小分子物质附着;TUNEL染色结果提示3 d与5 d组细胞凋亡数量接近,而7 d时细胞凋亡显著增加(P<0.001);PCR结果表明C-BMSC对ALP、COL-1、Runx2和OCN表达均高于细胞膜片(P<0.05);免疫荧光结果显示C-BMSC中ECM对ALP、COL-1、BSP表达均呈阳性;大鼠颅骨移植实验结果表明C-BMSC组骨体积与软组织体积比显著高于空白组。结论:本研究证明C-BMSC具有良好的成骨分化能力,这为骨缺损再生修复提供了一种新的策略。

Objective:To evaluate the bone defect regeneration and repair ability of human bone marrow mesenchymal stem cells/extracellular matrix complex(C-BMSC).Methods:C-BMSC was constructed in vitro and examined by gross observation,HE staining,and scanning electron microscope(SEM).TUNEL staining was used to detect the level of apoptosis.The expression level of osteogenic related factors was detected by immunofluorescence staining and PCR.The rat skull defect model was used to evaluate the bone defect regeneration and repair ability of C-BMSC in vivo.Results:Histomorphology examination showed that the extracellular matrix(ECM)of C-BMSC was the most abundant in the 5-day group.The ECM completely wrapped the cells in C-BMSC.The ECM fibers were strong and small molecular substances were attached.TUNEL staining result showed that the number of apoptosis in the 3-day and 5-day groups was close,but it was increased significantly for the cell apoptosis level at 7-day(P<0.001).PCR results showed that the expressions of ALP,COL-1,Runx2,and OCN in C-BMSC was higher than those in BMSC-CS(P<0.05).Immunofluorescence results showed that ECM in C-BMSC was positive for ALP,COL-1,and BSP.C-BMSC also showed remarkable ability of bone tissue regeneration and repair in vivo(P<0.05).Conclusion:This study proves that C-BMSC has outstanding osteogenic differentiation ability,which provides a new strategy for bone defect regeneration and repair.