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。
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。
Scheme 1. Concept illustration. The MnO2 NP-dotted hydrogel is fabricated and duly with MSCs for implantation after a serious long-span spinal cord transection 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.
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 indicated characteristic peaks in the products. d) Schematic illustration of HA hydrogels in the presence or absence 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.
Figure 2. In vitro evaluation of antioxidant effects of the MnO2 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. False-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 absence of cells. An initial concentration of H2O2 at 0.1 mM wasused in the culture medium without the presence of cells (b1) and the H2O2 levels were detected after incubation with the hydrogels for 1 h and 2 h (b2). Data in graph is presented as average ± SD (n=5). The significance between Blank and MnO2 groups at each time point is assessed 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 exposed to the ROS microenvironment simulating medium containing 0.1 mM H2O2. 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 pseudocolor (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 presented as average ± SD (n= 9 views). Representative images of the cellsin the blank (e2, e3) and MnO2 NP-dotted (e4, e5) hydrogels were presented, with n= 3 hydrogels for each group. Scale bar, 4 μm (a), 100 μm (d and e).
Figure 3. MSC-encapsulated hydrogels were implanted into rat spinal cord tissue after a longspan transection and antioxidant effects were detected after 7 days of implantation. a) dihydroethidium (DHE) staining was applied to directly reflect the ROS levels in the tissues. The corresponding representative micrographs (a1, a2) and histogram of the fluorescence intensity (a3) showed an obvious decrease of DHE distribution in the MnO2 group. b, c) Products of lipid peroxidation 4-hydroxynonenal (4-HNE) (b) and oxidative DNA damage 8-hydroxy-2'- deoxyguanosine (8-OHdG) (c) in the spinal cord tissues were detected, which confirmed the effective antioxidant and protective functions of the MnO2 nanoparticle (NP)-dotted hydrogel in vivo. Extent of oxidative damage was analyzed in terms of the percentage of the stained area with n = 6 micrographs (20 × objective magnification) of 3 animals for each group (b3, c3). Data represents mean ± SD. Statistical analysis among groups was preformed using two-tailed unpaired t-test. The double asterisk symbol (**) denotes to p< 0.001. Details were shown in boxes with dashed borders in representative images. Scale bar, 100 μm.
Figure 4. Motor functional recovery of rats after treatment of the hydrogels encapsulated with MSCs. a) Schematic illustration of the therapeutic experiment. b) Surgical procedure of longspan spinal cord transection and implantation of the MSCs-encapsulated hydrogels. The span of spinal cord transection was larger than the width of spinal cord tissue, with a length of 4 ± 0.5 mm compared to the width of 3 mm. c) Function recovery of animals on Day 28 post-surgery was evaluated through BBB scores. d) Analysis of % animal of BBB scores > 8 further indicated the function restoration in the presence of the MnO2 nanoparticle (NP)-dotted hydrogel containing MSCs. e, f) Typical records of animal walking gaits on Day 28 post-surgery showed direct evidence of the hindlimb walking patterns of the Blank group e) and the MnO2 group f). Significance assessment between two groups (Blank and MnO2 group, SCI and MnO2 group) was conducted using Mann–Whitney U test for data with normal distribution as well as variance homogeneity and two-tailed unpaired t-test for data with non-normal distribution. *p< 0.05, ** p< 0.001.
Figure 5. Investigation of nerve fiber regeneration across the lesion cavity on Day 28 postsurgery by labelling of neurofilament (NF, stained to red) and glial fibrillary acidic protein (GFAP, stained to green). a, b) Analysis of NF redistribution and GFAP+ astrocyte gathering in the areas adjacent to the lesion site. a) Glial scar formation was indicated by the gathered GFAP+ astrocytes in the spinal cord implanted with the blank hydrogel (a1), whereas implantation with the MnO2 nanoparticle (NP)-dotted hydrogel (a2) restrained the excessive gathering of the activated astrocytes and significantly induced NF regeneration across the lesion border. b) Redistribution of NF across the GFAP bordering was quantified in terms of % area of positive staining. c, d) Nerve fiber regeneration was analyzed by quantification of NF in sagittal sections.Redistribution of NF was analyzed in the lesion site, adjacent tissue and distant areas respectively of n = 10 micrographs for each group (c) and in representative sections for continuous distribution in the whole area as presented by continuous curves (d). e, f) Representative micrographs of the Blank (e) and MnO2 (f) groups showed the full-field situations of NF and GFAP expression (e1, f1) and the separated channel of NF distribution (e2, f2) in the spinal cord tissues, with detailed images magnified from the boxed adjacent (e3, f3) and distant (e4, f4) regions. Dashed lines denote to the lesion edges of interest. More and longer nerve fibers were observed in not only the lesion site and the adjacent tissue, but also the distant segments of the injured tissue in the MnO2 group than that in Blank group. Significance in (c) was assessed by Mann–Whitney U test, and the asterisk symbols (**) denotes to p< 0.001. Scale bar, 1 mm (e1, f1), 200 μm (e3, f3), 100 μm (e4, f4).
论文信息：
Liming Li, Bing Xiao, Jiafu Mu, Yu Zhang, Chenyang Zhang, Hongcui Cao, Rongjun Chen, Hirak Kumar Patra, Bo Yang, Shiqing Feng, Yasuhiko Tabata, Nigel K.H. Slater, Jianbin Tang,* Youqing Shen, and Jianqing Gao.* A MnO2 Nanoparticle-Dotted Hydrogel Promotes Spinal Cord Repair via Regulating Reactive Oxygen Species Microenvironment and Synergizing with Mesenchymal Stem Cells. ACS Nano 2019, DOI: 10.1021/acsnano.9b07598.
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