Liang Hong Sheng, Liang Peng, Xu Ye, Wu Jia Ning, Liang Tao, Xu Xiao Ping, Liu En Zhong
Department of Neurosurgery, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China.
Acta Neurochir (Wien). 2009 Sep;151(9):1113-20. doi: 10.1007/s00701-009-0322-5. Epub 2009 Apr 30.
Previous studies have shown that axonal outgrowth in the damaged central nervous system is closely related to the local microenvironment. Transplantation of bone marrow stromal cells (BMSC) or BMSC with some biomaterials has been used to treat various central nervous system diseases with some success. In the current study, we investigated if BMSC on denuded human amniotic membrane (DhAM) as a composite matrix could stimulate axonal outgrowth or not.
After completely removing the cells on the amniotic membrane with a tryptic and mechanical approach, we seeded BMSC on it. The MTS was applied to test the cytotoxicity of DhAM compared with PLGA and PLL. The morphology of the BMSC was observed by light, electronic and laser confocal microscopy. We also used four kinds of substance (PLL, DhAM, BMSC + PLL, BMSC + DhAM) to coculturing with the cortical neurons. Finally, the lengths of axons in each group were studied using the positive axon-specific marker NF-H.
The DhAM was devoid of cellular components and only its intact basement membrane was left. BMSC grew on the substrate and proliferated with a flat to fusiform morphology. In the MTS test, the results indicated that BMSC cultured in DhAM extract had a high survival rate (> 80%). Moreover, the cortical neural axons in the experimental group (BMSC + DhAM) were longer (287.37 +/- 12.72 microm) than in the other groups (P < 0.01).
This study demonstrates that the DhAM was a good carrier to support growth of BMSC and BMSC on DhAM was an effective composite matrix to support the outgrowth of the axons of rat cortical neurons in vitro. Future studies of the use of the composite matrix in disorders are planned.
先前的研究表明，受损中枢神经系统中的轴突生长与局部微环境密切相关。骨髓基质细胞（BMSC）或BMSC与某些生物材料的移植已被用于治疗各种中枢神经系统疾病，并取得了一定的成功。在本研究中，我们调查了以无细胞人羊膜（DhAM）作为复合基质的BMSC是否能刺激轴突生长。
用胰蛋白酶和机械方法完全去除羊膜上的细胞后，我们将BMSC接种其上。应用MTS法测试DhAM与聚乳酸-羟基乙酸共聚物（PLGA）和聚赖氨酸（PLL）相比的细胞毒性。通过光学、电子和激光共聚焦显微镜观察BMSC的形态。我们还使用四种物质（PLL、DhAM、BMSC + PLL、BMSC + DhAM）与皮质神经元共培养。最后，使用轴突特异性阳性标志物神经丝重链（NF-H）研究每组轴突的长度。
DhAM没有细胞成分，仅留下完整的基底膜。BMSC在基质上生长并以扁平至梭形形态增殖。在MTS测试中，结果表明在DhAM提取物中培养的BMSC具有较高的存活率（> 80%）。此外，实验组（BMSC + DhAM）的皮质神经轴突比其他组更长（287.37 +/- （12.72微米））（P < 0.01）。
本研究表明，DhAM是支持BMSC生长的良好载体，并且在DhAM上的BMSC是体外支持大鼠皮质神经元轴突生长的有效复合基质。计划在未来研究该复合基质在疾病中的应用。
