Madigan Nicolas N, Chen Bingkun K, Knight Andrew M, Rooney Gemma E, Sweeney Eva, Kinnavane Lisa, Yaszemski Michael J, Dockery Peter, O'Brien Timothy, McMahon Siobhan S, Windebank Anthony J
1 Department of Neurology, Mayo Clinic College of Medicine , Mayo Clinic, Rochester, Minnesota.
Tissue Eng Part A. 2014 Nov;20(21-22):2985-97. doi: 10.1089/ten.TEA.2013.0551. Epub 2014 Aug 11.
The use of multichannel polymer scaffolds in a complete spinal cord transection injury serves as a deconstructed model that allows for control of individual variables and direct observation of their effects on regeneration. In this study, scaffolds fabricated from positively charged oligo[poly(ethylene glycol)fumarate] (OPF(+)) hydrogel were implanted into rat spinal cords following T9 complete transection. OPF(+) scaffold channels were loaded with either syngeneic Schwann cells or mesenchymal stem cells derived from enhanced green fluorescent protein transgenic rats (eGFP-MSCs). Control scaffolds contained extracellular matrix only. The capacity of each scaffold type to influence the architecture of regenerated tissue after 4 weeks was examined by detailed immunohistochemistry and stereology. Astrocytosis was observed in a circumferential peripheral channel compartment. A structurally separate channel core contained scattered astrocytes, eGFP-MSCs, blood vessels, and regenerating axons. Cells double-staining with glial fibrillary acid protein (GFAP) and S-100 antibodies populated each scaffold type, demonstrating migration of an immature cell phenotype into the scaffold from the animal. eGFP-MSCs were distributed in close association with blood vessels. Axon regeneration was augmented by Schwann cell implantation, while eGFP-MSCs did not support axon growth. Methods of unbiased stereology provided physiologic estimates of blood vessel volume, length and surface area, mean vessel diameter, and cross-sectional area in each scaffold type. Schwann cell scaffolds had high numbers of small, densely packed vessels within the channels. eGFP-MSC scaffolds contained fewer, larger vessels. There was a positive linear correlation between axon counts and vessel length density, surface density, and volume fraction. Increased axon number also correlated with decreasing vessel diameter, implicating the importance of blood flow rate. Radial diffusion distances in vessels significantly correlated to axon number as a hyperbolic function, showing a need to engineer high numbers of small vessels in parallel to improving axonal densities. In conclusion, Schwann cells and eGFP-MSCs influenced the regenerating microenvironment with lasting effect on axonal and blood vessel growth. OPF(+) scaffolds in a complete transection model allowed for a detailed comparative, histologic analysis of the cellular architecture in response to each cell type and provided insight into physiologic characteristics that may support axon regeneration.
在完全性脊髓横断损伤中使用多通道聚合物支架作为一种解构模型，可控制各个变量并直接观察它们对再生的影响。在本研究中，将由带正电荷的聚（乙二醇富马酸酯）寡聚物（OPF(+)）水凝胶制成的支架植入T9完全横断后的大鼠脊髓中。OPF(+)支架通道中接种了同基因雪旺细胞或源自增强型绿色荧光蛋白转基因大鼠的间充质干细胞（eGFP-MSCs）。对照支架仅包含细胞外基质。通过详细的免疫组织化学和体视学检查了每种支架类型在4周后影响再生组织结构的能力。在周围环形通道隔室中观察到星形胶质细胞增生。结构上独立的通道核心包含散在的星形胶质细胞、eGFP-MSCs、血管和再生轴突。用胶质纤维酸性蛋白（GFAP）和S-100抗体进行双重染色的细胞存在于每种支架类型中，表明未成熟细胞表型从动物迁移到支架中。eGFP-MSCs与血管紧密分布在一起。雪旺细胞植入增强了轴突再生，而eGFP-MSCs不支持轴突生长。无偏体视学方法提供了每种支架类型中血管体积、长度和表面积、平均血管直径和横截面积的生理学估计值。雪旺细胞支架在通道内有大量小而密集排列的血管。eGFP-MSC支架中的血管较少且较大。轴突计数与血管长度密度、表面密度和体积分数之间存在正线性相关性。轴突数量增加也与血管直径减小相关，这暗示了血流速度的重要性。血管中的径向扩散距离与轴突数量呈双曲线函数显著相关，表明需要并行构建大量小血管以提高轴突密度。总之，雪旺细胞和eGFP-MSCs影响了再生微环境，对轴突和血管生长产生持久影响。完全横断模型中的OPF(+)支架允许对每种细胞类型响应下的细胞结构进行详细的比较组织学分析，并深入了解可能支持轴突再生的生理学特征。
