Pizzute Tyler, Zhang Ying, He Fan, Pei Ming
Department of Orthopaedics, Stem Cell and Tissue Engineering Laboratory, West Virginia University, Morgantown, WV, USA. Exercise Physiology, West Virginia University, Morgantown, WV, USA. Equal contribution to this work.
Biomed Mater. 2016 Aug 10;11(4):045009. doi: 10.1088/1748-6041/11/4/045009.
Developing an in vitro microenvironment using cell-derived decellularized extracellular matrix (dECM) is a promising approach to efficiently expand adult stem cells for cartilage engineering and regeneration. Ascorbic acid serves as a critical stimulus for cells to synthesize collagens, which constitute the major component of dECM. In this study, we hypothesized that optimization of ascorbate treatment would maximize the rejuvenation effect of dECM on expanded stem cells from human infrapatellar fat pad in both proliferation and chondrogenic differentiation. In the duration regimen study, we found that dECM without L-ascorbic acid phosphate (AA) treatment, exhibiting lower stiffness measured by atomic force microscopy, yielded expanded cells with higher proliferation capacity but lower chondrogenic potential when compared to those with varied durations of AA treatment. dECM with 250 µM of AA treatment for 10 d had better rejuvenation in chondrogenic capacity if the deposited cells were from passage 2 rather than passage 5, despite no significant difference in matrix stiffness. In the dose regimen study, we found that dECMs deposited by varied concentrations of AA yielded expanded cells with higher proliferation capacity despite lower expression levels of stem cell related surface markers. Compared to cells expanded on tissue culture polystyrene, those on dECM exhibited greater chondrogenic potential, particularly for the dECMs with 50 µM and 250 µM of AA treatment. With the supplementation of ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor targeting procollagen synthesis, the dECM with 50 µM of AA treatment exhibited a dramatic decrease in the rejuvenation effect of expanded cell chondrogenic potential at both mRNA and protein levels despite no significant difference in matrix stiffness. Defined AA treatments during matrix preparation will benefit dECM-mediated stem cell engineering and future treatments for cartilage defects.
利用细胞衍生的脱细胞细胞外基质（dECM）构建体外微环境是一种很有前景的方法，可有效扩增成体干细胞用于软骨工程和再生。抗坏血酸是细胞合成胶原蛋白的关键刺激物，而胶原蛋白是dECM的主要成分。在本研究中，我们假设优化抗坏血酸盐处理将使dECM对来自人髌下脂肪垫的扩增干细胞在增殖和成软骨分化方面的年轻化作用最大化。在持续时间方案研究中，我们发现未经L - 抗坏血酸磷酸酯（AA）处理的dECM，通过原子力显微镜测量显示出较低的硬度，与经过不同持续时间AA处理的dECM相比，其扩增的细胞具有更高的增殖能力，但成软骨潜力较低。如果接种的细胞来自第2代而非第5代，用250 μM AA处理10天的dECM在成软骨能力方面具有更好的年轻化效果，尽管基质硬度没有显著差异。在剂量方案研究中，我们发现不同浓度AA沉积的dECM产生的扩增细胞具有更高的增殖能力，尽管干细胞相关表面标志物的表达水平较低。与在组织培养聚苯乙烯上扩增的细胞相比，在dECM上的细胞表现出更大的成软骨潜力，特别是对于用50 μM和250 μM AA处理的dECM。通过添加针对前胶原合成的抑制剂3,4 - 二羟基苯甲酸乙酯（EDHB），用50 μM AA处理的dECM在mRNA和蛋白质水平上扩增细胞成软骨潜力的年轻化效果均显著降低，尽管基质硬度没有显著差异。在基质制备过程中确定的AA处理将有利于dECM介导的干细胞工程以及未来软骨缺损的治疗。
