Bian Zhengjun, Sun Jianhua
Department of Orthopedics, The First Affiliated Hospital, Shihezi University College of Medicine Shihezi 832008, China.
Int J Clin Exp Pathol. 2015 Feb 1;8(2):1093-103. eCollection 2015.
To develop tissue engineering scaffolds consisting of self-assembling KLD-12 polypeptide/TGF-β1 nanofiber gel, for the induction of mesenchymal stem cell (MSCs) differentiation into nucleus pulposus (NP)-like cells.
The release of TGF-β1 from KLD-12 polypeptide gels containing varying TGF-β1 concentrations was detected by ELISA. MSCs were isolated with a density gradient method and their differentiation into NP-like cells was analyzed in KLD-12 polypeptide/TGF-β1- or KLD-12 polypeptide control nanofiber-gel 3D-cultures. The Alcianblue method, Real-time quantitative PCR (RT-qPCR), and immunocytochemistry were used to measure the expression of extracellular matrix (ECM) molecules, such as aggrecan, glycosaminoglycans (GAGs), and type II collagen.
ELISA results documented favorable time-dependent release characteristics of TGF-β1 in the KLD-12 polypeptide/TGF-β1 gel scaffolds. The results of CCK-8 cell proliferation assay showed the TGF-β1 containing scaffolds induced higher growth rate in MSCs compared to the control group. Calcein-AM/PI fluorescent staining showed: the cells in the gel grew well, maintaining the circular shape of cells, and the spindle and fusiform shape of cells on the gel edges. The cell viability displayed a survival rate of 89.14% ± 2.468 for the TGF-β1 group with no significant difference between the two groups at 14 d of culture. The production of ECM was monitored showing higher expression of GAGs in the TGF-β1 group (P < 0.01) with highest amounts at 10 d and 14 d compared to 4 d and 7 d (P < 0.05). Real-time PCR results revealed that the expression levels of collagen II and aggrecan mRNA were higher in the TGF-β1 group (P < 0.05). Finally, immunocytochemical staining of collagen II confirmed the higher expression levels.
A scaffold containing a KLD-12 polypeptide/TGF-β1-nanofiber gel and MSCs differentiated into NP-like cells is able to produce ECM and has the potential to serve as a three-dimensional (3-D) support scaffold for the filling of early postoperative residual cavities and the treatment of intervertebral disc degeneration.
构建由自组装KLD-12多肽/TGF-β1纳米纤维凝胶组成的组织工程支架，用于诱导间充质干细胞（MSCs）分化为髓核（NP）样细胞。
采用ELISA法检测不同TGF-β1浓度的KLD-12多肽凝胶中TGF-β1的释放情况。采用密度梯度法分离MSCs，并在KLD-12多肽/TGF-β1或KLD-12多肽对照纳米纤维凝胶三维培养体系中分析其向NP样细胞的分化情况。采用阿尔辛蓝法、实时定量PCR（RT-qPCR）和免疫细胞化学法检测细胞外基质（ECM）分子，如聚集蛋白聚糖、糖胺聚糖（GAGs）和Ⅱ型胶原的表达。
ELISA结果表明TGF-β1在KLD-12多肽/TGF-β1凝胶支架中具有良好的时间依赖性释放特性。CCK-8细胞增殖试验结果显示，与对照组相比，含TGF-β1的支架可诱导MSCs更高的生长速率。钙黄绿素-AM/PI荧光染色显示：凝胶中的细胞生长良好，保持细胞的圆形形态，以及凝胶边缘细胞的纺锤形和梭形形态。在培养14天时TGF-β1组细胞活力显示存活率为89.14%±2.468，两组之间无显著差异。监测ECM的产生，结果显示TGF-β1组GAGs表达较高（P<0.01），与4天和7天相比，在10天和14天时含量最高（P<0.05）。实时PCR结果显示，TGF-β1组中Ⅱ型胶原和聚集蛋白聚糖mRNA的表达水平较高（P<0.05）。最后，Ⅱ型胶原的免疫细胞化学染色证实了其较高的表达水平。
含有KLD-12多肽/TGF-β1纳米纤维凝胶的支架以及分化为NP样细胞的MSCs能够产生ECM，并且有潜力作为三维（3-D）支撑支架用于填充术后早期残留腔隙以及治疗椎间盘退变。
