骨髓间充质干细胞在多孔胶原海绵支架中的增殖及成腱分化
Proliferation and tenogenic differentiation of bone marrow mesenchymal stem cells in a porous collagen sponge scaffold.
作者信息
Zhang Bing-Yu, Xu Pu, Luo Qing, Song Guan-Bin
机构信息
Department of College of Bioinformatics, Chongqing University of Posts and Telecommunications, Chongqing 400065, China.
Department of College of Bioengineering, Chongqing University, Chongqing 400030, China.
出版信息
World J Stem Cells. 2021 Jan 26;13(1):115-127. doi: 10.4252/wjsc.v13.i1.115.
BACKGROUND
Collagen is one of the most commonly used natural biomaterials for tendon tissue engineering. One of the possible practical ways to further enhance tendon repair is to combine a porous collagen sponge scaffold with a suitable growth factor or cytokine that has an inherent ability to promote the recruitment, proliferation, and tenogenic differentiation of cells. However, there is an incomplete understanding of which growth factors are sufficient and optimal for the tenogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in a collagen sponge-based 3D culture system.
AIM
To identify one or more ideal growth factors that benefit the proliferation and tenogenic differentiation of rat BMSCs in a porous collagen sponge scaffold.
METHODS
We constructed a 3D culture system based on a type I collagen sponge scaffold. The surface topography of the collagen sponge scaffold was observed by scanning electron microscopy. Primary BMSCs were isolated from Sprague-Dawley rats. Cell survival on the surfaces of the scaffolds with different growth factors was assessed by live/dead assay and CCK-8 assay. The mRNA and protein expression levels were confirmed by quantitative real-time polymerase chain reaction and Western blot, respectively. The deposited collagen was assessed by Sirius Red staining.
RESULTS
Transforming growth factor β1 (TGF-β1) showed great promise in the tenogenic differentiation of BMSCs compared to growth differentiation factor 7 (GDF-7) and insulin-like growth factor 1 (IGF-1) in both the 2D and 3D cultures, and the 3D culture enhanced the differentiation of BMSCs into tenocytes well beyond the level of induction in the 2D culture after TGF-β1 treatment. In the 2D culture, the proliferation of the BMSCs showed no significant changes compared to the control group after TGF-β1, IGF-1, or GDF-7 treatment. However, TGF-β1 and GDF-7 could increase the cell proliferation in the 3D culture. Strangely, we also found more dead cells in the BMSC-collagen sponge constructs that were treated with TGF-β1. Moreover, TGF-β1 promoted more collagen deposition in both the 2D and 3D cultures.
CONCLUSION
Collagen sponge-based 3D culture with TGF-β1 enhances the responsiveness of the proliferation and tenogenic differentiation of rat BMSCs.
背景
胶原蛋白是肌腱组织工程中最常用的天然生物材料之一。进一步增强肌腱修复的一种可能的实际方法是将多孔胶原蛋白海绵支架与具有促进细胞募集、增殖和向肌腱细胞分化固有能力的合适生长因子或细胞因子相结合。然而，对于在基于胶原蛋白海绵的三维培养系统中，哪些生长因子足以促进大鼠骨髓间充质干细胞（BMSCs）向肌腱细胞分化且效果最佳，目前尚不完全清楚。
目的
确定一种或多种有利于大鼠BMSCs在多孔胶原蛋白海绵支架中增殖和向肌腱细胞分化的理想生长因子。
方法
构建基于I型胶原蛋白海绵支架的三维培养系统。通过扫描电子显微镜观察胶原蛋白海绵支架的表面形貌。从Sprague-Dawley大鼠中分离出原代BMSCs。通过活/死检测和CCK-8检测评估不同生长因子处理下支架表面的细胞存活率。分别通过定量实时聚合酶链反应和蛋白质印迹法确认mRNA和蛋白质表达水平。通过天狼星红染色评估沉积的胶原蛋白。
结果
在二维和三维培养中，与生长分化因子7（GDF-7）和胰岛素样生长因子1（IGF-1）相比，转化生长因子β1（TGF-β1）在BMSCs向肌腱细胞分化方面显示出巨大潜力，并且在TGF-β1处理后，三维培养比二维培养更能显著增强BMSCs向肌腱细胞的分化。在二维培养中，TGF-β1、IGF-1或GDF-7处理后，BMSCs的增殖与对照组相比无显著变化。然而，TGF-β1和GDF-7可增加三维培养中的细胞增殖。奇怪的是，我们还发现在用TGF-β1处理的BMSC-胶原蛋白海绵构建体中有更多死细胞。此外，TGF-β1在二维和三维培养中均促进更多胶原蛋白沉积。
结论
基于胶原蛋白海绵的三维培养与TGF-β1可增强大鼠BMSCs增殖和向肌腱细胞分化的反应性。
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