含有二氧化锰纳米粒子的水凝胶促进脊髓修复
创伤性的脊髓损伤（SCI）是衰竭性损伤之一。由于脊髓损伤后自身恢复能力差以及中枢神经系统极度敏感，因此神经组织修复面临着巨大挑战。临床上通常采用保守治疗的手段治疗脊髓损伤，但是神经功能却无法恢复。
衍变干细胞和生物材料支架移植可有望治疗脊髓损伤。植入的干细胞，如间充质干细胞可补偿受损的神经细胞及营养，而支架可为植入的细胞提供细胞外基质（ECM）并促进组织再生。透明质酸（HA）是从天然的ECM中衍生出的聚多糖，HA可抑制神经胶质疤痕的形成，因而利于脊髓组织的修复。HA水凝胶具有多孔结构，并且其粘弹性与天然脊髓组织相似。更为重要的是，HA支架在脊髓半切损伤中具有一定的神经保护作用。由于HA缺乏细胞粘附性，因而其桥接作用受到很大限制。在先前的工作中，作者制备的层粘连蛋白衍生肽PPFLMLLKGSTR可显著促进干细胞黏附生长及神经组织桥接，因此作者用其修饰HA水凝胶（HA-peptide），改善HA水凝胶的细胞粘附性。
由于水凝胶支架中产生活性氧自由基（ROS）以及脊髓损伤造成的炎症反应，会导致载细胞水凝胶中的干细胞发生凋亡及功能紊乱，最终导致脊髓损伤疗效较差。为此，作者向HA-pep
tide水凝胶中引入二氧化锰纳米粒子（MnO2 NPs），利用MnO2可分解双氧水（H2大学生创业基金O打往天堂的电话2）产生氧气（O2）的特性，改善氧化应激环境，从而有效提高干细胞的生存率（Scheme 1）。
Scheme 1. Concept illustration. The MnO2 NP-dotted hydrogel is fabricated and duly with MSCs for implantation after a rious long-span spinal cord tranction with a lesion gap of 4 ± 0.5 mm. The MnO2 NP improves the survival, integration and neural differentiation of the transplanted MSCs and promotes nerve tissue regeneration via mitigating the oxidant microenvironment.朱俊伟
通过傅立叶转换红外谱图（FTIR）及1H NMR可看出，作者成功将PPFLMLLKGSTR多肽键接到HA链上，MnO2 NPs的加入使得HA水凝胶具有较强的磁共振显影性能。此外，HA水凝胶展现出3D多孔结构。
Figure 1. Characterization of materials and hydrogels. a) FTIR detection of aldehyde-modified HA chains (HA-CHO). b, c) 1H NMR detection of HA-adipic dihydrazide (HA-ADH) (b) and peptide modified aldehyde-HA chains (HA-peptide) (c). Red arrows indicate
d characteristic peaks in the products. d) Schematic illustration of HA hydrogels in the prence or abnce of MnO2 nanoparticles (NPs). e) The blank and MnO2 NP-dotted hydrogels were imaged by MRI, which detected signals of MnO2 NPs. f, g) Scanning electron microscopy (SEM) micrographs of blank (f) and MnO2 NP-dotted (g) hydrogel scaffolds showing highly porous 3D structures. h) XPS examination of the MnO2 NP-dotted hydrogel confirmed successful encapsulation of MnO2 NPs in the hydrogel. Scale bar, 100 μm.
为了检测水凝胶的生物相容性，作者将人胎盘羊膜间充质干细胞在水凝胶中进行3D培养，结果发现，含有MnO2 NPs的水凝胶对细胞的粘附形态没有影响，表明MnO2 NPs的加入没有影响水凝胶的生物相容性。作者将水凝胶浸入含有H2O2的氧化介质中检测水凝胶的抗氧化能力，结果发现，含有MnO2 NPs的水凝胶可有效降低介质中的H2O2水平。接着将载有细胞的水凝胶浸入氧化介质中，对细胞内的ROS水平进行检测，含有MnO2 NPs的水凝胶中的细胞ROS水平明显低于水凝胶组，表明水凝胶加载MnO2 NPs后具有非常优异的抗氧化功能。进一步通过活死染色检测水凝胶中的细胞生存情况，结果发现，含有MnO2 NPs的水凝胶中只有少数的死细胞，表明MnO2 NPs的加入可有效改善氧化应激环境，减
少细胞死亡。体内实验同样表明含有MnO2 NPs的水凝胶具有良好的抗氧化性能，可用于神经损伤修复。
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Figure 2. In vitro evaluation of antioxidant effects of the MnO2开元小学 nanoparticle (NP) dotted hydrogel. a) Scanning electron microscopy (SEM) micrographs showing similar adhesive morphologies of MSCs in the blank (a1) and MnO2 NP-dotted (a2) hydrogels. Fal-color images showed more detailed views of the boxed areas. b) Evaluation of in vitro antioxidant effects of the blank and MnO2 NP-dotted hydrogels through simulating pathological ROS microenvironment in the abnce of cells. An initial concentration of H2O2 at 0.1 mM wasud in the culture medium without the prence of cells (b1) and the H2O2 容易英文levels were detected after incubation with the hydrogels for 1 h and 2 h (b2). Data in graph is prented as average ± SD (n=5). The significance between Blank and MnO2 groups at each time point is assd by unpairedt-test (two-tailed value), and the double asterisk symbol (**) denotes to p< 0.001. c-e) Protective effects of the hydrogels to 3D cultured MSCs were investigated in a simulated in vitro ROS microenvironment. c) MSCs were encapsulated in the hydrogels and allowed to adhere overnight before being expod to the ROS microenvironment simulating medium containing 0.1 mM H2O2白色狗狗种类. d) After 24 h, the intracellular ROS levels of MSCs were quantified (d1) in the blank (d2, d3)
and MnO2 NP-dotted (d4, d5) hydrogels after Dichloro-dihydro-fluorescein diacetate (DCFH-DA) labelling (n=3), demonstrating effective antioxidant impact of the MnO2 NP-dotted hydrogel for theMSCs. Green, DCFH-DA; blue, DAPI. Fluorescence of DCFH-DA in (d2, d4) were analyzed using pudocolor (d3, d5) according to mean intensities to further show more detailed differences. e) Cytoviability of the MSCs after 24 h of in cubation was analyzed by Live/Dead assay. Numbers of live cells (green) anddead cells (red) were quantified in terms of stained areas (e1). Data in graphis prented as average ± SD (n= 9 views). Reprentative images of the cellsin the blank (e2, e3) and MnO2 NP-dotted (e4, e5) hydrogels were prented, with n= 3 hydrogels for each group. Scale bar, 4 μm (a), 100 μm (d and e).
